CN103403000A - Solid forms of gyrase inhibitor (R)-1-ethyl-3-[6-fluoro-5-[2-(1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl]-7-(tetrahydrofuran-2-yl)-1h-benzimidazol-2-yl]urea - Google Patents

Abstract

The present application is directed to solid forms of compounds of formula (I) and pharmaceutically acceptable salts thereof that inhibit bacterial gyrase and/or Topo IV, and pharmaceutical compositions comprising said compounds and salts. These compounds and salts are useful in treating bacterial infections.

Description

The gyrase inhibitor of solid form (R)-fluoro-5-[2-of 1-ethyl-3-[6-(1-hydroxyl-1-methyl-ethyl) pyrimidine-5-yl]-7-(tetrahydrofuran (THF)-2-yl)-1 hydrogen-benzimidazolyl-2 radicals-yl] urea

Cross reference with related application

The application requires in the interests of the U.S. Provisional Patent Application sequence number 61/433,169 of submission on January 14th, 2011 according to 35U.S.C. § 119, and its complete content is integrated with this paper by reference.

The application background

Have recognized that for a long time the resistibility of bacterial antibiotic, and it is regarded as serious whole world health problem in today.Due to resistibility, some bacteriums infect or are difficult to antibiotic therapy or or even can't treat.Immediate development along with the multi-medicine resistance in the specific bacteria bacterial strain, it is especially serious that this problem has become, described specific bacteria bacterial strain for example streptococcus pneumoniae (Streptococcus pneumoniae) (SP), mycobacterium tuberculosis (Mycobacterium tuberculosis) and faecalis (Enterococcus).Vancomycin resistance enterococcal appearance is alarming especially, because vancomycin was the unique effective microbiotic that is used for the treatment of this infection in the past, and for many infection, is regarded as the medicine of " last resort ".Although many other anti-medicine bacteriums for example faecalis do not cause life-threatening disease, but the gene that exists such worry and be induction of resistance may spread to more fatal organism, described organism is streptococcus aureus (Staphylococcus aureus) for example, generally popular (the De Clerq of its X-1497 resistance, Deng the people, Current Opinion in Anti-infective Investigational Drugs, 1999,1,1; Levy, " The Challenge of Antibiotic Resistance ", Scientific American, March, 1998).

Another concern is can how to spread rapidly antibiotic resistance.For example, until nineteen sixties, SP is generally to the penicillin sensitivity, and in 1987, the U.S. only 0.02% SP bacterial strain resistance is arranged.Yet, to nineteen ninety-five, it is reported that SP is approximately 7 percent to the resistance of penicillin, and in the some parts area of the U.S. up to 30%(Lewis, FDA Consumer magazine(1995 September); Gershman in The Medical Reporter, 1997).

Particularly the center of formation and the propagation of anti-medicine organism has been served as by hospital.The infection that occurs in hospital is called nosocomial infection, becomes more and more serious problem.In annual 2000000 Americans that infect of hospital, these surpass at least a microbiotic of half opposing in infecting.The Center for Disease Control report, in 1992, surpass 13,000 inpatients and die from bacterium infection (Lewis, " the The Rise of Antibiotic-Resistant Infections " that antibiotic therapy is had to resistance, FDA Consumer magazine, September nineteen ninety-five).

Due to the needs with anti-medicine bacterium antagonism and the increasing failure of available medicine, the interest to the recovery of exploitation new antibiotic has appearred.A kind of attractive strategy for the exploitation new antibiotic is to suppress DNA gyrase and/or topoisomerase I V, and these bacterial enzymes are essential to DNA replication dna and to bacterial cell growth and division, are therefore that institute is essential.Gyrase and/or topoisomerase I V also to DNA, transcribe, repair and recombinate in event relevant.

Gyrase is one of topoisomerase, topoisomerase be the topological isomer change of catalytic dna one group of enzyme (generally referring to, Kornberg and Baker, DNA Replication, the 2nd edition, the 12nd chapter, 1992, W.H.Freeman and Co.; Drlica, Molecular Microbiology, 1992,6,425; Drlica and Zhao, Microbiology and Molecular Biology Reviews, 1997,61,377-392 page).Gyrase self is controlled the DNA superhelix and alleviate the topological pressure that occurs when the DNA of parental duplex chain is untied during reproduction process.The gyrase also lax closed annular duplex DNA of catalysis is converted to for the more favourable negative supercoiling form of restructuring.The mechanism of superhelix reaction relates to the parcel of gyrase around the zone of DNA, and the double-strand break in that zone, make second zone of DNA pass fracture, and reconnect the chain of fracture.This type of mechanism of splitting is the feature of II type topoisomerase.The superhelix reaction drives by the combination of ATP and gyrase.ATP is hydrolyzed subsequently in reaction process.This ATP combination and follow-up hydrolysis cause the conformational change in the gyrase of DNA combination, and this is that it is active essential.It has also been found that the horizontal dependency ATP of DNA superhelix (or lax)/ADP ratio.In the situation that there is not ATP, the gyrase super coiled DNA that only can relax.

The protein tetramer of 400 kilodaltons that the DNA of bacteria gyrase is comprised of two A subunits (GyrA) and two B subunits (GyrB).The combination of DNA with split relevant to GyrA, and ATP by the GyrB protein bound and the hydrolysis.GyrB forms by the N-terminal structural domain with atpase activity with GyrA and the interactional C-terminal structural domain of DNA.By contrast, eucaryon II type topoisomerase is homodimer, its can relax negative supercoiling and positive supercoiling but can not introduce negative supercoiling.Ideally, based on the microbiotic of anti-bacteria DNA gyrase and/or topoisomerase I V, for this kind of enzyme, be selectively, and be relative non-activity for eucaryon II type topoisomerase.

Topoisomerase I V mainly eliminates the karyomit(e) dimer that connects when DNA replication dna finishes.

Widely used quinolone antibiotic anti-bacteria DNA gyrase (GyrA) and/or topoisomerase I V(ParC).The example of quinolone comprises early stage compound for example Nalidixic Acid and oxolinic acid, and more effective fluoroquinolone of later stage for example norfloxicin, Ciprofloxacin and trovafloxacin.These compounds are combined with GyrA and/or ParC, and the stable mixture that splits, thereby suppress overall gyrase function, cause necrocytosis.Fluoroquinolone suppresses gyrase (GyrA) and/or topoisomerase I V(ParC) catalytic subunit (referring to Drlica and Zhao, Microbiology and Molecular Biology Reviews, 1997,61,377-392).Yet resistance also has been considered to the problem (WHO Report, " Use of Quinolones in Food Animals and Potential Impact on Human Health ", 1998) about this compounds.For quinolone, the same with the microbiotic of other kinds, the common fast development of bacterium that is exposed to more early stage compound goes out the crossed resistance for more effective compound in similar.

Being responsible for be hydrolyzed the catalysis of supplying enzyme by ATP, to have enough to meet the need/reset the relevant subunit of institute's energy requirement be respectively the GyrB(gyrase) and ParE(topoisomerase I V) (referring to Champoux, J.J., Annu.Rev.Biochem., 2001,70,369-413 page).It will be useful (referring to people such as Charifson, J.Med.Chem., 2008,51,5243-5263 page) that the compound of these the identical ATP-binding site in target GyrB and ParE subunit infects for the treatment various bacteria.

There is the less known inhibitor of being combined with GyrB.Example comprises tonka bean camphor, Vulkamycin. PA-93 and coumermycinA1, cyclothialidine, cinodine and PR 1350.Tonka bean camphor has shown very closely is combined with GyrB.For example, Vulkamycin. PA-93 forms hydrogen bond network with described albumen and several hydrophobicity contacts.Although Vulkamycin. PA-93 and ATP seem combination in ATP-binding site really, in the bonding position of two kinds of compounds, exist MIN overlapping.Lap is the sugared unit of Vulkamycin. PA-93 and the VITAMIN B4 of ATP (Maxwell, Trends in Microbiology, 1997,5,102).

For the tonka bean camphor tolerant bacteria, the most general point mutation is on surperficial arginine residues, and it is combined (Arg136 in intestinal bacteria (E.coli) GyrB) with the carbonyl of coumarin ring.Although have the enzyme of this sudden change, show lower superhelix and atpase activity, they are also more insensitive (Maxwell, Mol.Microbiol., 1993,9,681) to the inhibition by the tonka bean camphor medicine.

Although be the supercoiled effective inhibitor of gyrase, tonka bean camphor is not widely used as microbiotic yet.Low penetration power due to it in bacterium, eukaryote toxicity and bad water-soluble, they be generally inappropriate (Maxwell, Trends in Microbiology, 1997,5,102).To wish new effective GyrB and ParE inhibitor, its overcome these shortcomings and preferably do not rely on Arg136 in conjunction with for activity.This type of inhibitor will be attractive microbiotic material standed for, not perplex the history of the antibiotic resistance problem of other kinds.

Because bacterium has become important public health problem for antibiotic resistibility, so exist exploitation to upgrade and stronger antibiotic lasting needs.More specifically, there are the antibiotic needs that before are not used for the treatment of the new species compound of bacterium infection for representative.Target GyrB(gyrase) and ParE(topoisomerase I V) compound of ATP-binding site in subunit infects for the treatment various bacteria will be useful.This compounds will be useful especially for the nosocomial infection in treatment hospital, and wherein the formation of tolerant bacteria and propagation become more and more general.

Application outlined

The application relates to the fluoro-5-[2-(1-hydroxyl of solid form (R)-1-ethyl-3-[6--1-methyl-ethyl) pyrimidine-5-yl]-7-(tetrahydrofuran (THF)-2-yl)-1 hydrogen-benzimidazolyl-2 radicals-yl] urea (" 6 fluoro-benzimidazolyl-carbamide compound ").In one embodiment, the application provides the solid form I of described 6-fluorobenzene imidazolyl carbamide compound, it is characterized in that X-ray powder diffraction pattern (XPRD) is when using Cu K _αDuring radiation measurement, comprise at least 3 approximate peak positions (2 θ angles ± 0.2), when the Er Xitas (2 θ) of XPRD from about 5 degree to 38 degree collected, it was selected from 9.3,11.7,12.1,12.4,14.5,15.9,16.3,16.6,18.5,19.4,21.5,22.3,22.8,23.8,24.5,25.7,28.1,28.4,30.3 and 33.4.Its feature of solid form I also can be as used Cu K _αThe X-ray powder diffraction pattern that is substantially similar to Fig. 1 that radiation is measured and have starting temperature in the about endotherm(ic)peak of 318 ℃, it is by measuring with the about differential scanning calorimetries of 10 ℃ of/minute scanning temperature.The application also provides the method for the preparation of 6-fluorobenzene the imidazolyl carbamide compound of crystallized form I, by the free alkali by solid material, is suspended in solvent systems separate solid, and described solvent systems comprises alcohol and ether.

Another embodiment of the application provides the solid form II of the hydrochloride of described 6-fluorobenzene imidazolyl carbamide compound, it is characterized in that X-ray powder diffraction pattern (XPRD) is when using Cu K _αDuring radiation measurement, comprise at least 3 approximate peak positions (2 θ angles ± 0.2), when XPRD from about 5 degree when approximately 2 θ of 38 degree collect, it is selected from 6.7,9.2,16.7,18.6,19.5,20.5,25.6 and 27.5.Its feature of solid form II also can be as used Cu K _αThe X-ray powder diffraction pattern that is substantially similar to Fig. 4 that radiation is measured and have starting temperature in the about endotherm(ic)peak of 252 ℃, it is by measuring with the about differential scanning calorimetries of 10 ℃ of/minute scanning temperature.The solid form II of the hydrochloride of 6-fluorobenzene imidazolyl carbamide compound can be suspended in by the free alkali by 6-fluorobenzene imidazolyl carbamide compound the acid solvent mixture that comprises one or more ether solvents and water.

Further embodiment in the application is the unbodied form III of 6-fluorobenzene imidazolyl carbamide compound (free alkali), it is characterized in that having can not the identification diffraction peak broad halo be the use Cu K of feature _αThe X-ray powder diffraction pattern (XPRD) of radiation.The application's further embodiment also provides the method for the preparation of the unbodied form III of 6-fluorobenzene imidazolyl carbamide compound (free alkali), and it comprises the mutual dry 6-fluorobenzene of freeze-drying, spraying drying, roller drying or pulse imidazolyl carbamide compound solution.

Another embodiment of the application also provides the unbodied form IV of the mesylate of described 6-fluorobenzene imidazolyl carbamide compound, it is characterized in that having can not the identification diffraction peak broad halo be the use Cu K of feature _αThe X-ray powder diffraction pattern (XPRD) of radiation.

The accompanying drawing summary

Fig. 1 has shown the X-ray powder diffraction pattern of the solid form I of 6-fluorobenzene imidazolyl carbamide compound (free alkali), and it is from approximately extremely approximately 38 degree 2 θ collections of 5 degree.

Fig. 2 has shown the DSC(differential scanning calorimetry measurement of the solid form I of 6-fluorobenzene imidazolyl carbamide compound (free alkali)) thermogram.

Fig. 3 has shown the TGA(thermogravimetric analysis of the solid form I of 6-fluorobenzene imidazolyl carbamide compound (free alkali)) thermogram.

Fig. 4 has shown the X-ray powder diffraction pattern of solid form II of the hydrochloride of 6-fluorobenzene imidazolyl carbamide compound.

Fig. 5 has shown the DSC thermogram of solid form II of the hydrochloride of 6-fluorobenzene imidazolyl carbamide compound.

Fig. 6 has shown the TGA thermogram of the solid form II of 6-fluorobenzene imidazolyl carbamide compound.

Fig. 7 is the X-ray powder diffraction pattern of the amorphous solid III of 6-fluorobenzene imidazolyl carbamide compound (free alkali).

Fig. 8 has shown that it is then three larger exotherms that the DSC thermogram of the amorphous solid III of 6-fluorobenzene imidazolyl carbamide compound (free alkali) presents a little exotherm.

Fig. 9 is the X-ray powder diffraction pattern of amorphous solid IV of the mesylate of 6-fluorobenzene imidazolyl carbamide compound.

Figure 10 is the mesylate of 6-fluorobenzene imidazolyl carbamide compound ¹The H-NMR wave spectrum.

Describe in detail

The application relates to the fluoro-5-[2-(1-hydroxyl of (R)-1-ethyl-3-[6-of novel pure solid form basically-1-methyl-ethyl) pyrimidine-5-yl]-7-(tetrahydrofuran (THF)-2-yl)-1 hydrogen-benzimidazolyl-2 radicals-yl] urea (" 6-fluorobenzene imidazolyl carbamide compound ").

The contriver has found the crystalline free base form of described compound (form I), pharmacologically acceptable salts (the form II of 6-fluorobenzene imidazolyl carbamide compound, corresponding hydrochloride) crystallized form, the unbodied form of described free alkali (form III), and the unbodied form of the mesylate of described compound (form IV).

Thus, the application novel solid form I that aspect is 6-fluorobenzene imidazolyl carbamide compound (free alkali).In one aspect, the application provides the method for the preparation of the solid form I of 6-fluorobenzene imidazolyl carbamide compound.

Basically the solid form I of pure 6-fluorobenzene imidazolyl carbamide compound can be prepared by the compound of amorphous or crystallization, by making this compound, with the solvent systems that comprises alcohol and ether, contacts and separate solid.By in envrionment temperature, making the 6-fluorobenzene in solvent and imidazolyl carbamide compound solution is saturated and allow mixture section of standing time expand (for example spending the night), 6-fluorobenzene imidazolyl carbamide compound can contact with solvent.Alternatively, 6-fluorobenzene imidazolyl carbamide compound are cooled to solution room temperature or lower and separate solid form I in can under the rising temperature is for example refluxing, being dissolved in solvent subsequently.

In an embodiment of described method, basically the solid form I of pure 6-fluorobenzene imidazolyl carbamide compound can be prepared by the compound of amorphous or crystallized form, by in room temperature, preparing the saturated solution of compound in suitable solvent and separating the form I that produces.In practice, this can make and when allowing solution to be cooled to room temperature, obtain saturated solution by in rising temperature (until reflux) is dissolved in solvent by the 6-fluorobenzene of q.s imidazolyl carbamide compound, completing, and therefrom be precipitated out also can be separated for form I.In one embodiment, can be from reaction mixture, separating 6-fluorobenzene imidazolyl carbamide compound by revising the solvability of compound in solvent.For example, it is out precipitable that the temperature of removing part or all of solvent or reducing mixture can reduce solvent borne and the form I of 6-fluorobenzene imidazolyl carbamide compound.Alternatively, to adding the precipitable solid form I that goes out described compound of the second solvent in mixture.

In one embodiment, being used for the solvent of precipitation forms I is the mixture of ethanol and ether.Separate the solid that generates form I is provided.

The solid form I of 6-fluorobenzene imidazolyl carbamide compound can identify by following characteristics: as the differential scanning calorimetry by using 10 ℃ of/minute scanning speeds, measure, one at the about wide endothermic curve of 250 ℃, has extrapolated onset at the about melting endothermic curve of 318 ℃; Basically the X-ray powder diffraction pattern as shown in table 1 and Fig. 1, wherein the XRPD pattern uses the powder diffractometer that is equipped with Cu X-ray tube wave source to measure.With Cu K α ₁Radiation is irradiated sample and from approximately 5 to about 2 θ of 40 ° collecting the XRPD data.The relative intensity that one skilled in the art will realize that the XPRD peak can significantly change, it depends on the direction that is subjected to the sample product and instrument type and the setting of use, it is illustrational that thereby the intensity in the included XPRD spike of this paper is come to a certain extent, not is intended to for absolute comparison.

Fig. 1 is the X-ray powder diffraction pattern of the solid form I of the described 6-fluorobenzene collected from about 5 degree to about 40 degree 2 θ imidazolyl carbamide compound (free alkali).Corresponding to the peak that has more than or equal to the X-ray powder diffraction pattern of 5% relative intensity, in table 1, list.

Fig. 2 has shown the DSC thermogram of the solid form I of described 6-fluorobenzene imidazolyl carbamide compound, its present have initial transition approximately 250 ℃ wide endothermic curve and have initial transition at the about endothermic curve of 318 ℃.The peak and the starting temperature that one skilled in the art will realize that endothermic curve can rely on experiment condition and change.The 2.5mg sample of described solid, about 35 ℃ of balances approximately 10 minutes, is collected to the data in Fig. 2.In sample collection phase process, temperature is with the approximately speed increase of 10 ℃/minute.

Fig. 3 is the TGA(thermogravimetric analysis of the solid form I of described 6-fluorobenzene imidazolyl carbamide compound) thermogram, it presents in 50 to 300 ℃ of temperature ranges approximately the starting weight of 15% per-cent and reduces and follow between 300 to 400 ℃ more 25% extra weight to reduce.

In one embodiment, the invention provides the solidification form I of the described compound of chemical formula (I):

In another embodiment, solid form I is characterised in that X-ray powder diffraction pattern (XPRD) is when using Cu K _αDuring radiation measurement, comprise at least 3 approximate peak positions (2 θ angles ± 0.2), when 2 θs of XPRD from about 5 degree to 38 degree collected, it was selected from 9.3,11.7,12.1,12.4,14.5,15.9,16.3,16.6,18.5,19.4,21.5,22.3,22.8,23.8,24.5,25.7,28.1,28.4,30.3 and 33.4.

In another embodiment, solid form I is characterised in that X-ray powder diffraction pattern (XPRD) is when using Cu K _αDuring radiation measurement, comprise at least 3 approximate peak positions (2 θ angles ± 0.2), when 2 θs of XPRD from about 5 degree to 38 degree collected, it was selected from 9.3,16.6,18.5,19.4,21.5 and 25.7.

In another embodiment, solid form I is characterised in that as used Cu K _αRadiometric, be substantially similar to the X-ray powder diffraction pattern of Fig. 1.

In another embodiment, it is further characterized in that solid form I as by what with the about differential scanning calorimetry of 10 ℃ of/minute scanning temperature, measure, has starting temperature in the about endotherm(ic)peak of 318 ℃.

In another embodiment, the invention provides the method for the preparation of the crystallized form I of the described compound of formula (I), it comprises and is suspended in the free alkali of solid material in the solvent systems that comprises alcohol and ether and separates described solid.

In another embodiment, solid form I can keep stablizing at least one month at 40 ℃ with relative humidity up to 75% time.

The XRPD pattern peak of the solid form I of table 1.6-fluorobenzene imidazolyl carbamide compound

In yet another aspect, the application provides the crystallized form II of the salt acid salt of described 6-fluorobenzene imidazolyl carbamide compound.In one embodiment, the application provides the method for the preparation of the solid form II of described 6-fluorobenzene imidazolyl carbamide compound.The salt acid salt of the acceptable 6-fluorobenzene of pharmacy imidazolyl carbamide compound can be prepared by any method known to those skilled in the art.

In some embodiments, the salt acid salt of described 6-fluorobenzene imidazolyl carbamide compound is by forming in the solution that acid is added to described compound and being precipitated out thus.In other embodiments, by revising the solubleness of salt in solvent, can be from reaction mixture, separating described acid salt.For example, remove part or all of solvent or reduce solubleness and the salt that mixture temperature can reduce the hydrochloride of 6-fluorobenzene imidazolyl carbamide compound and be precipitated out.Alternatively, the second solvent is added in mixture and can precipitate described salt.

In further embodiment, gaseous hydrochloric acid can bubbling by the solution of 6-fluorobenzene imidazolyl carbamide compound, until single acid salt of compound is produced out.In specific embodiment, the hydrochloric acid of stoichiometric quantity and 6-fluorobenzene imidazolyl carbamide compound can mix to form single salt acid salt of compound.For example, 6-fluorobenzene the solution of imidazolyl carbamide compound in polar solvent and the combined of stoichiometric quantity.Example applicable to the polar solvent of the solid form II of precipitation 6-fluorobenzene imidazolyl carbamide compound hydrochloride comprises ether, for example ether and tetrahydrofuran (THF) (THF).

In a specific embodiment, the 6-fluorobenzene in THF of stoichiometric quantity imidazolyl carbamide compound and water hydrochloric acid mix lentamente, and in the stirring at room mixture overnight.The 6-fluorobenzene of solid white imidazolyl carbamide compound hydrochloride are precipitated out.Separate solid, and wash with water with vacuum under dry.

The solid form II shape of described 6-fluorobenzene imidazolyl carbamide compound can be identified by following characteristics: as the differential scanning calorimetry by using 10 ℃ of/minute scanning speeds, measure, one at the about wide endothermic curve of 210 ℃, has the extrapolation starting point at the about melting endothermic curve of 252 ℃; Basically the X-ray powder diffraction pattern as shown in table 2 and Fig. 4, wherein the XRPD pattern uses the powder diffractometer that is equipped with Cu X-ray tube wave source to measure.With Cu K α ₁Radiation is irradiated sample and from approximately 5 to about 2 θ of 40 ° collecting the XRPD data.The direction that the relative intensity that one skilled in the art will realize that the XRPD peak can depend on sample significantly changes.

Fig. 4 is the X-ray powder diffraction pattern of solid form II of the hydrochloride of the described 6-fluorobenzene collected from about 5 degree to about 38 degree 2 θ imidazolyl carbamide compound.Corresponding to the peak that has more than or equal to the X-ray powder diffraction pattern of 5% relative intensity, in table 2, list.

Fig. 5 has shown the DSC thermogram of solid form II of the hydrochloride of described 6-fluorobenzene imidazolyl carbamide compound, its present have approximately 210 ℃ endothermic curve and at the about endothermic curve of 252 ℃.The peak and the starting temperature that one skilled in the art will realize that endothermic curve can rely on experiment condition and change.The 1mg sample of solid form, about 35 ℃ of balances approximately 10 minutes, is collected to the data in Fig. 5.In sample collection phase process, temperature is with the approximately speed increase of 10 ℃/minute.

Fig. 6 is the TGA thermogram of solid form II of the hydrochloride of described 6-fluorobenzene imidazolyl carbamide compound, it presents the approximately initial weight of 8% per-cent minimizing between 100-220 ℃, second weight followed by approximately 8% per-cent extra between 240-270 ℃ reduces, followed by between 270-300 ℃ approximately the 3rd weight of 3% per-cent reduce.One skilled in the art will realize that the starting temperature that weight reduces can rely on experiment condition and change.Although the applicant does not wish to stick to the specific explanations that the endothermic curve in DSC and the weight in TGA are reduced, the transition that seems to have large peak in DSC is because the weight by in TGA reduces the melting transition that is associated with material degradation that implies.

In one embodiment, the invention provides the hydrochloride of the compound of formula (I):

In another embodiment, hydrochloride is solid form II.

In another embodiment, the solid form II of hydrochloride is characterised in that X-ray powder diffraction pattern (XPRD) is when using Cu K _αDuring radiation measurement, comprise at least 3 approximate peak positions (2 θ angles ± 0.2), when XPRD from about 5 degree when approximately 2 θ of 38 degree collect, it is selected from 6.7,9.2,16.7,18.6,19.5,20.5,25.6 and 27.5.

In another embodiment, the solid form II of hydrochloride is characterised in that as used CuK _αRadiometric, be substantially similar to the X-ray powder diffraction pattern of Fig. 4.

In another embodiment, it is further characterized in that the solid form II of hydrochloride as by what with the about differential scanning calorimetry of 10 ℃ of/minute scanning temperature, measure, has starting temperature in the about endotherm(ic)peak of 252 ℃.

Also in another embodiment, the invention provides the method for the preparation of the solid form II of the hydrochloride of the described compound of formula (I), it comprises the free alkali of 6-fluorobenzene imidazolyl carbamide compound is suspended in the acid solvent mixture that comprises more than one ethers and water.

In another embodiment, the solid form II of hydrochloride can keep stablizing at least one month at 40 ℃ with relative humidity up to 75% time.

The XRPD pattern peak of the solid form II of table 2.6-fluorobenzene imidazolyl carbamide compound

Another aspect of the application provides the composition that comprises unbodied 6-fluorobenzene imidazolyl carbamide compound (free alkali).For 6-fluorobenzene imidazolyl carbamide compound or its salt term " amorphous " used herein, refer to solid shape form, wherein 6-fluorobenzene imidazolyl urea molecule generally exist and do not form cognizable lattice or crystal unit cell with unordered arranging.When it was carried out to X-ray powder diffraction, complete unbodied compound did not produce the feature diffraction pattern of crystallized form.The X-ray powder diffraction of local unbodied material may still lack the feature diffraction pattern of crystalline form, to such an extent as to because observe from the too weak noise that can not surpass in derivative peak of the crystallising part of sample.Fig. 7 has shown the X-ray powder diffraction pattern of the unbodied form III of described 6-fluorobenzene imidazolyl carbamide compound (free alkali).

Fig. 8 has shown the DSC thermogram of the unbodied form III of described 6-fluorobenzene imidazolyl carbamide compound (free alkali), and it presents a little exotherm is then 3 larger exotherms.Little exotherm has the starting temperature of 127 ℃ and described three exotherms have the starting temperature of 183 ℃, 226 ℃, 279 ℃.The peak and the starting temperature that one skilled in the art will realize that exotherm and endothermic curve can rely on experiment condition and change.The 2.9mg sample of described unbodied 6-fluorobenzene imidazolyl carbamide compound, about 35 ℃ of balances approximately 10 minutes, is collected to the data in Fig. 8.In sample collection phase process, temperature is with the approximately speed increase of 10 ℃/minute.

In another embodiment, the invention provides the described fluorobenzene of formula I the unbodied form III of imidazolyl carbamide compound:

In another embodiment, the unbodied form III of described fluorobenzene imidazolyl carbamide compound, it is characterized in that having can not the identification diffraction peak broad halo be the use Cu K of feature _αThe X-ray powder diffraction pattern (XPRD) of radiation.

Also in another embodiment, the invention provides the method for the preparation of the unbodied form III of described 6-fluorobenzene imidazolyl carbamide compound, it comprises the mutual dry 6-fluorobenzene of freeze-drying, spraying drying, roller drying or pulse imidazolyl carbamide compound solution.

In yet another aspect, the application provides the form IV of amorphous solid phase of the mesylate of described 6-fluorobenzene imidazolyl carbamide compound.In one embodiment, the application provides the method for the preparation of the form IV of the mesylate of 6-fluorobenzene imidazolyl carbamide compound.The mesylate of the acceptable 6-fluorobenzene of pharmacy imidazolyl carbamide compound can be prepared by any method known to those skilled in the art.For example, the solution of methanesulfonic can be added in the solution of 6-fluorobenzene imidazolyl carbamide compound, until prepare single acid salt of described compound.In one embodiment, the mesylate of 6-fluorobenzene imidazolyl carbamide compound is by forming in the solution that acid is added to 6-fluorobenzene imidazolyl carbamide compound and being precipitated out thus.In other embodiments, by revising the solubleness of salt in solvent, can be from reaction mixture, separating described acid salt.For example, remove part or all of solvent or reduce solubleness and the salt that mixture temperature can reduce the mesylate of 6-fluorobenzene imidazolyl carbamide compound and be precipitated out.Therefore, in some embodiments, after precipitated solvent or from solution, by evaporation section solvent (for example, using rotatory evaporator) concentrated solution, collecting amorphous material afterwards.Alternatively, the second solvent is added in mixture and can precipitate described salt.

The mesylate of described 6-fluorobenzene imidazolyl carbamide compound can use any method well known by persons skilled in the art to be converted to amorphous solid.Unbodied 6-fluorobenzene imidazolyl carbamide compound mesylate are characterised in that the diffraction pattern that does not have crystallized form.The X-ray powder diffraction of local unbodied 6-fluorobenzene imidazolyl carbamide compound mesylate may still lack the characteristic attribute of crystalline form, to such an extent as to because observe from the too weak noise that can not surpass in derivative peak of the crystallising part of sample.Fig. 9 is the X-ray powder diffraction pattern of unbodied form IV of the mesylate of described 6-fluorobenzene imidazolyl carbamide compound.

In one embodiment, the amorphous mesylate of described 6-fluorobenzene imidazolyl carbamide compound can be prepared by the described salts solution in suitable solvent by spraying drying.Spraying drying is well-known in the art, and the pharmacy medicine for example of the material through being usually used in the dry heat sensitivity.Spraying drying also provides the consistent particle distribution that can reproduce quite well.Any gas may be used to dried powder, although usually use air.If material to air-sensitive, can use rare gas element for example nitrogen or argon so.Solution, slurry, suspension or the milk sap of conversion salt can be suitable for preparing the solid amorphous form IV of the mesylate of described 6-fluorobenzene imidazolyl carbamide compound with any method that produces pressed powder.For example, lyophilize, drum drying or pulses switch drying can be for generation of the amorphous mesylates of described 6-fluorobenzene imidazolyl carbamide compound.

In one embodiment, use equipped condenser receive the 6-fluorobenzene of the sprayable drying of spraying (nanospray) moisture eliminator in polar solvent the solution of imidazolyl carbamide compound.Temperature in can remain on 80-120 ℃.

In another embodiment, the invention provides the unbodied form IV of the mesylate of the 6-fluorobenzene of formula I imidazolyl carbamide compound.

In another embodiment, the unbodied form IV of the mesylate of described 6-fluorobenzene imidazolyl carbamide compound, it is characterized in that having can not the identification diffraction peak broad halo be the use Cu K of feature _αThe X-ray powder diffraction pattern (XPRD) of radiation.

Be appreciated that solid form I and II and be respectively the 6-fluorobenzene and unsetting solid form III and the IV of the free alkali of imidazolyl carbamide compound and mesylate, except having XRPD as herein described, DSC, TGA and other feature, also can have other features of not describing, such as but not limited to having water or more than one solvent molecule.

X-ray powder diffraction pattern (XRPD): use the Bruker D8Discover system (Bruker AXS, Madison, WI) that is equipped with sealed tube wave source and Hi-Star area detector, record the XRPD pattern of crystallized form in room temperature with reflective-mode.X ray generator is with 40kV tension force and 35mA current practice.Powdered sample is placed on Si zero background wafer.With each time shutter of 120 seconds registration two frames.Data are integrated on 3 ° of-41 ° of 2 scopes with the step-length of 0.02 ° subsequently, and are merged into a continuous pattern.

X-ray powder diffraction pattern (XRPD) about amorphous form: use Bruker D8Advance system (the Bruker AXS that is equipped with the Vantec-1 position-sensitive detector, Madison, WI), in room temperature, with reflective-mode, record the XRPD pattern of amorphous solid.X ray generator is with 40kV tension force and 45mA current practice.Powdered sample is placed on Si zero background fixer, uses and in experimentation, with 15rpm, rotate with continuous mode at the variable gap on detector.With 0.0144653 degree increment (0.25 second/step) from 3 to 40 degree, collect data.

Differential scanning calorimetry (DSC): use DSC Q2000 differential scanning calorimeter (TA Instruments, New Castle, DE) to carry out DSC to the sample of material.This instrument is calibrated with indium.About 1-2mg samples weighing is covered in the aluminium dish of crimping to the lid with free of pinholes or pin hole.The DSC sample also scans from 30 ℃ to the temperature shown in drawing with 50mL/ minute nitrogen stream with the heating rate of 10 ℃/minute.Regulating DSC(MDSC) descend the sample of operation to be conditioned 1 ℃ of+He – in every 60 seconds, its ramp rate (ramp rates) is 2 or 3 ℃/minute.

Data are collected by Thermal Advantage Q SeriesTM software and are analyzed by Universal Analysis2000 software (TA Instruments, New Castle, DE).

Thermogravimetric analysis (TGA): model Q5000 thermogravimetric analyzer (TA Instruments, New Castle, DE) is measured for TGA.Usually, the sample that about 3-5mg is heavy scans from 30 ℃ to the temperature shown in drawing with the heating rate of 10 ℃/minute.Data are collected by Thermal Advantage Q SeriesTM software and are analyzed by Universal Analysis2000 software (TA Instruments, New Castle, DE).

The present invention also provides pharmaceutical composition, and it comprises compound or its pharmacologically acceptable salts of formula (I), and pharmaceutically acceptable carrier, adjuvant or vehicle.

The present invention also provides control, treatment or has reduced in patient's hospital or the method for progress, seriousness or the effect of bacterium infection in non-hospital, and it comprises to described patient uses the compound that comprises formula (I) or the pharmaceutical composition of its pharmacologically acceptable salts.

in another embodiment, the present invention also provides control, the progress that in treatment or minimizing patient's hospital or non-hospital, bacterium infects, the method of seriousness or effect, it comprises to described patient uses the compound that comprises formula (I) or the pharmaceutical composition of its pharmacologically acceptable salts, and wherein said bacterium infects one or more the existence that is characterised in that in following: streptococcus pneumoniae, staphylococcus epidermidis (Staphylococcus epidermidis), enterococcus faecalis (Enterococcus faecalis), streptococcus aureus (Staphylococcus aureus), clostridium difficile (Clostridium difficile), moraxelle catarrhalis (Moraxella catarrhalis), neisseria gonorrhoeae (Neisseria gonorrhoeae), Neisseria meningitidis (Neisseria meningitidis), the multiple disease (Mycobacterium avium complex) of mycobacterium avium, mycobacterium abscessus (Mycobacterium abscessus), mycobacterium kansasii (Mycobacterium kansasii), mycobacterium buruli (Mycobacterium ulcerans), Chlamydophila pneumoniae (Chlamydophila pneumoniae), chlamydia trachomatis (Chlamydia trachomatis), hemophilus influenzae (Haemophilus influenzae), micrococcus scarlatinae (Streptococcus pyogenes), or beta hemolytic streptococcus (β-haemolytic streptococci).

in another embodiment, the present invention also provides control, the progress that in hospital in treatment or minimizing patient or non-hospital, bacterium infects, the method of seriousness or effect, it comprises to described patient uses the compound that comprises formula (I) or the pharmaceutical composition of its pharmacologically acceptable salts, and wherein said bacterium infects and is selected from one or more in following: upper respiratory tract infection, lower respiratory infection, ear infection, pleura lung and bronchial infection, concurrent urinary tract infection, non-concurrent urinary tract infection, intra-abdominal infection, cardiovascular infection, bloodstream infection, septicemia, microbemia, CNS infects, Skin and soft tissue infection, GI infects, Arthropyosis infection, genital infection, ocular infection or granuloma infect, non-concurrent skin and skin texture infect (uSSSI), concurrent skin and skin texture infect (cSSSI), catheter infections, pharyngitis, sinusitis, external otitis, otitis media, bronchitis, pyothorax, pneumonia, Community-acquired bacterial pneumonia (CABP), Nosocomial Pneumonia (HAP), the Nosocomial bacterial pneumonia, ventilator associated pneumonia (VAP), infection in diabetic foot, vancomycin resistance faecalis infects, urocystitis and pyelonephritis, urinary stone disease, prostatitis, peritonitis, concurrent intra-abdominal infection (cIAI) and other intra-abdominal infection, the dialysis Related peritonitis, the internal organ abscess, endocarditis, myocarditis, pericarditis, the relevant septicemia of transfusion, meningitis, encephalitis, cerebral abscess, osteomyelitis, sacroiliitis, genital ulcer, urethritis, vaginitis, trachelitis, gingivitis, conjunctivitis, keratitis, endophthalmitis, infection in cystic fibrosis patient or heat generation neutrophilic granulocyte reduce disease patient's infection.

In another embodiment, bacterium infects and to be selected from one or more in following: Community-acquired bacterial pneumonia (CABP), Nosocomial Pneumonia (HAP), Nosocomial bacterial pneumonia, ventilator associated pneumonia (VAP), microbemia, infection in diabetic foot, catheter infections, non-concurrent skin and skin texture infect that (uSSSI), concurrent skin and skin texture infect (cSSSI), vancomycin resistance faecalis infects or osteomyelitis.

According to another embodiment, the invention provides the method that reduces or suppress the bacterial number in biological sample.This method comprises makes described biological sample contact with compound or its pharmacologically acceptable salts of formula (I).

Term " biological sample " comprises cell culture or its extract as used herein; Derive from mammiferous biopsy material or its extract; With blood, saliva, urine, ight soil, seminal fluid, tear or other body fluid or its extract.Term " biological sample " also comprises live organism, " compound of the present invention is contacted " with biological sample in said case and the term synonym that " described compound or the composition that comprises described compound is applied to Mammals ".

Gyrase of the present invention and/or topoisomerase I V inhibitor or its pharmaceutical salts can be mixed with pharmaceutical composition be used to being applied to Mammals or people.Another embodiment of the invention is, effectively treats or prevent the described pharmaceutical composition that bacterium infects, and it comprises gyrase and/or topoisomerase I V inhibitor and the pharmaceutically acceptable carrier of the amount that is enough to reduce bacterial number with measuring.As used herein, term " can reduce bacterial number with measuring " and means at the sample that contains described inhibitor and only contain measurable variation of the number of bacteria between germy sample.

According to another embodiment, described method of the present invention is useful to the patient in the treatment veterinary applications, includes but not limited to zoological park, laboratory, mankind partner and farm-animals, comprises primate, rodent, Reptilia and birds.The example of described animal includes but not limited to cavy, hamster, gerbil jird, rat, mouse, rabbit, dog, cat, horse, pig, sheep, ox, goat, deer, rhesus monkey, monkey, thin,tough silk hair monkey (tamarind), man like ape, baboon, gorilla, chimpanzee, orangutan, gibbon, ostrich, chicken, turkey, duck and goose.

Term " non-nosocomial infection " also is called Community Acquired Infections.

In another embodiment, bacterium infects one or more the existence be characterised in that in streptococcus pneumoniae, enterococcus faecalis or streptococcus aureus.

In another embodiment, bacterium infects one or more the existence be characterised in that in intestinal bacteria, moraxelle catarrhalis or hemophilus influenzae.

In another embodiment, the bacterium infection characteristic is one or more the existence in following: clostridium difficile, neisseria gonorrhoeae, Neisseria meningitidis, the multiple disease of mycobacterium avium, mycobacterium abscessus, mycobacterium kansasii, mycobacterium buruli, Chlamydophila pneumoniae and chlamydia trachomatis.

In another embodiment, bacterium infects one or more the existence be characterised in that in following: streptococcus pneumoniae, staphylococcus epidermidis, enterococcus faecalis, streptococcus aureus, clostridium difficile, moraxelle catarrhalis, neisseria gonorrhoeae, Neisseria meningitidis, the multiple disease of mycobacterium avium, mycobacterium abscessus, mycobacterium kansasii, mycobacterium buruli, Chlamydophila pneumoniae, chlamydia trachomatis, hemophilus influenzae, micrococcus scarlatinae or beta hemolytic streptococcus.

in some embodiments, bacterium infects one or more the existence be characterised in that in following: X-1497 resistance streptococcus aureus, fluoroquinolone resistance streptococcus aureus, vancomycin intermediate resistance streptococcus aureus, Linezolid resistance streptococcus aureus, the penicillin resistance streptococcus pneumoniae, macrolide resistance streptococcus pneumoniae, fluoroquinolone resistance streptococcus pneumoniae, vancomycin resistance enterococcus faecalis, Linezolid resistance enterococcus faecalis, fluoroquinolone resistance enterococcus faecalis, vancomycin resistance faecium (Enterococcus faecium), Linezolid resistance faecium, fluoroquinolone resistance faecium, Ampicillin Trihydrate resistance faecium, macrolide resistance hemophilus influenzae, beta-lactam resistance hemophilus influenzae, fluoroquinolone resistance hemophilus influenzae, beta-lactam resistance moraxelle catarrhalis, X-1497 resistance staphylococcus epidermidis, X-1497 resistance staphylococcus epidermidis, vancomycin resistance staphylococcus epidermidis, fluoroquinolone resistance staphylococcus epidermidis, macrolide resistance mycoplasma pneumoniae, vazadrine resistance mycobacterium tuberculosis, the rifampicin resistance mycobacterium tuberculosis, X-1497 resistance coagulase negative staphylococcus (Coagulase negative staphylococcus), fluoroquinolone resistance coagulase negative staphylococcus, glycopeptide intermediate resistance streptococcus aureus, vancomycin resistance streptococcus aureus, heterogeneous vancomycin intermediate resistance streptococcus aureus, heterogeneous vancomycin resistance streptococcus aureus, macrolide-lincosamide-streptogramin resistance staphylococcus (Staphylococcus), beta-lactam resistance enterococcus faecalis, beta-lactam resistance faecium, ketone lactone resistance streptococcus pneumoniae, ketone lactone resistance micrococcus scarlatinae, macrolide resistance micrococcus scarlatinae, vancomycin resistance staphylococcus epidermidis, fluoroquinolone resistance neisseria gonorrhoeae, multi-medicine resistance Pseudomonas aeruginosa (Pseudomonas aeruginosa) or cynnematin resistance neisseria gonorrhoeae.

According to another embodiment, X-1497 resistance staphylococcus is selected from X-1497 resistance streptococcus aureus, X-1497 resistance staphylococcus epidermidis or X-1497 resistance coagulase negative staphylococcus.

In some embodiments, the form of the compound of formula (I) or its pharmacologically acceptable salts is used for the treatment of Community-acquired MRSA (that is, cMRSA).

In other embodiments, a kind of form of the compound of formula (I) or its pharmacologically acceptable salts are used for the treatment of daptomycin resistance organism, include but not limited to daptomycin resistance faecium and daptomycin resistance streptococcus aureus.

According to another embodiment, fluoroquinolone resistance staphylococcus is selected from fluoroquinolone resistance streptococcus aureus, fluoroquinolone resistance staphylococcus epidermidis or fluoroquinolone resistance coagulase negative staphylococcus.

According to another embodiment, glycopeptide resistance staphylococcus is selected from glycopeptide intermediate resistance streptococcus aureus, vancomycin resistance streptococcus aureus, vancomycin intermediate resistance streptococcus aureus, heterogeneous vancomycin intermediate resistance streptococcus aureus or heterogeneous vancomycin resistance streptococcus aureus.

According to another embodiment, macrolide-lincosamide-streptogramin resistance staphylococcus is macrolide-lincosamide-streptogramin resistance streptococcus aureus.

According to another embodiment, Linezolid resistance faecalis is selected from Linezolid resistance enterococcus faecalis or Linezolid resistance faecium.

According to another embodiment, glycopeptide resistance faecalis is selected from vancomycin resistance faecium or vancomycin resistance enterococcus faecalis.

According to another embodiment, beta-lactam resistance enterococcus faecalis is beta-lactam resistance faecium.

According to another embodiment, the penicillin resistance suis is the penicillin resistance streptococcus pneumoniae.

According to another embodiment, macrolide resistance suis is macrolide resistance streptococcus pneumoniae

According to another embodiment, ketone lactone resistance suis is selected from macrolide resistance streptococcus pneumoniae and ketone lactone resistance micrococcus scarlatinae.

According to another embodiment, fluoroquinolone resistance suis is fluoroquinolone resistance streptococcus pneumoniae.

According to another embodiment, beta-lactam resistance influenzae is beta-lactam resistance hemophilus influenzae.

According to another embodiment, fluoroquinolone resistance influenzae is fluoroquinolone resistance hemophilus influenzae.

According to another embodiment, macrolide resistance influenzae is macrolide resistance hemophilus influenzae.

According to another embodiment, macrolide resistance mycoplasma is macrolide resistance mycoplasma pneumoniae.

According to another embodiment, vazadrine resistance mycobacterium is vazadrine resistance mycobacterium tuberculosis.

According to another embodiment, the rifampicin resistance mycobacterium is the rifampicin resistance mycobacterium tuberculosis.

According to another embodiment, beta-lactam resistance catarrhalis is beta-lactam resistance moraxelle catarrhalis.

according to another embodiment, bacterium infects one or more the existence be characterised in that in following: X-1497 resistance streptococcus aureus, fluoroquinolone resistance streptococcus aureus, vancomycin intermediate resistance streptococcus aureus, Linezolid resistance streptococcus aureus, the penicillin resistance streptococcus pneumoniae, macrolide resistance streptococcus pneumoniae, fluoroquinolone resistance streptococcus pneumoniae, vancomycin resistance enterococcus faecalis, Linezolid resistance enterococcus faecalis, fluoroquinolone resistance enterococcus faecalis, vancomycin resistance faecium, Linezolid resistance faecium, fluoroquinolone resistance faecium, Ampicillin Trihydrate resistance faecium, macrolide resistance hemophilus influenzae, beta-lactam resistance hemophilus influenzae, fluoroquinolone resistance hemophilus influenzae, beta-lactam resistance moraxelle catarrhalis, X-1497 resistance staphylococcus epidermidis, X-1497 resistance staphylococcus epidermidis, vancomycin resistance staphylococcus epidermidis, fluoroquinolone resistance staphylococcus epidermidis, macrolide resistance mycoplasma pneumoniae, vazadrine resistance mycobacterium tuberculosis, the rifampicin resistance mycobacterium tuberculosis, fluoroquinolone resistance neisseria gonorrhoeae or cynnematin resistance neisseria gonorrhoeae.

according to another embodiment, bacterium infects one or more the existence be characterised in that in following: X-1497 resistance streptococcus aureus, X-1497 resistance staphylococcus epidermidis, X-1497 resistance coagulase negative staphylococcus, fluoroquinolone resistance streptococcus aureus, fluoroquinolone resistance staphylococcus epidermidis, fluoroquinolone resistance coagulase negative staphylococcus, vancomycin resistance streptococcus aureus, glycopeptide intermediate resistance streptococcus aureus, vancomycin resistance streptococcus aureus, vancomycin intermediate resistance streptococcus aureus, heterogeneous vancomycin intermediate resistance streptococcus aureus, heterogeneous vancomycin resistance streptococcus aureus, vancomycin resistance faecium, vancomycin resistance enterococcus faecalis, the penicillin resistance streptococcus pneumoniae, macrolide resistance streptococcus pneumoniae, fluoroquinolone resistance streptococcus pneumoniae, macrolide resistance micrococcus scarlatinae or beta-lactam resistance hemophilus influenzae.

According to another embodiment, bacterium infects one or more the existence be characterised in that in following: X-1497 resistance streptococcus aureus, vancomycin resistance faecium, vancomycin resistance enterococcus faecalis, vancomycin resistance streptococcus aureus, vancomycin intermediate resistance streptococcus aureus, heterogeneous vancomycin intermediate resistance streptococcus aureus, heterogeneous vancomycin resistance streptococcus aureus, multi-medicine resistance Pseudomonas aeruginosa, vazadrine resistance mycobacterium tuberculosis and rifampicin resistance mycobacterium tuberculosis.

The pharmacologically acceptable salts of compound of the present invention comprises derived from those of the acceptable inorganic and organic bronsted lowry acids and bases bronsted lowry of pharmacy.the example of suitable hydrochlorate comprises acetate, adipate, alginate, aspartate, benzoate, benzene sulfonate, hydrosulfate, butyrates, Citrate trianion, camphorate, camsilate, cyclopentane propionate, digluconate, dodecyl sulfate, esilate, formate, fumarate, glucose enanthate (glucoheptanoate), glycerophosphate, oxyacetate, Hemisulphate (hemisulfate), enanthate, hexanoate, hydrochloride, hydrobromide, hydriodide, the 2-isethionate, lactic acid salt, maleate, malonate, mesylate, the 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, pectate, persulphate, 3-phenylpropionic acid salt, phosphoric acid salt, picrate, pivalate, propionic salt, salicylate, succinate, vitriol, tartrate, thiocyanate-, tosylate and undecylate.Other acid are oxalic acid for example, although self not that pharmacy is acceptable, can be for the preparation of salt, described salt is used as intermediate in obtaining compound of the present invention and the acceptable acid salt of pharmacy thereof.

Salt derived from suitable alkali comprises basic metal (for example sodium and potassium), alkaline-earth metal (for example magnesium), ammonium and N ⁺(C _1-4Alkyl) ₄Salt.The present invention has also imagined any alkaline nitrogen-containing group quaternized of compound disclosed herein.Water or oil soluble or dispersible product can pass through this type of quaternized acquisition.

Pharmaceutical composition of the present invention comprises compound or its pharmacologically acceptable salts and the pharmaceutically acceptable carrier of formula (I).Such composition selectively comprises other therapeutical agent.This type of reagent includes but not limited to microbiotic, anti-inflammatory agent, matrix metallo-proteinase inhibitor, lipoxidase inhibitor, cytokine antagonist, immunosuppressor, carcinostatic agent, antiviral agent, cytokine, somatomedin, immunomodulator, prostaglandin(PG) or anti-angiogenic hyper-proliferative compound.

Term " pharmaceutically acceptable carrier " refers to be applied to the patient and not destroy the non-toxic carrier of its pharmacological activity together with compound of the present invention.

can include but not limited to ion-exchanger for the pharmaceutically acceptable carrier of pharmaceutical composition of the present invention, aluminium, aluminum stearate, Yelkin TTS, serum protein are human serum albumin for example, buffer substance is phosphoric acid salt for example, glycine, Sorbic Acid, potassium sorbate, the partial glyceride mixture of saturated vegetable fatty acid, water, salt or ionogen, Protamine sulfates for example, Sodium phosphate dibasic, potassium hydrogen phosphate, sodium-chlor, zinc salt, colloid silicon, Magnesium Trisilicate, polyvinylpyrrolidone, based on cellulosic material, polyoxyethylene glycol, Xylo-Mucine, polyacrylate, wax, polyethylene-polyoxypropylene-block polymer, lanolin and self-emulsifying drug delivery systems (SEDDS) be alpha-tocopherol for example, cetomacrogol 1000 succinate or other similar polymerization delivery matrices.

Term " pharmacy effective dose " refers to effectively measure in the bacterium infection in treatment or in improving the patient.Term " prevention significant quantity " refers to the effective amount that the bacterium in preventing or basically alleviating the patient infects.

Rely on concrete symptom or morbid state to be treated or that prevent, usually be applied to treatment or prevent that the other therapeutical agent of this symptom from can use together with described inhibitor of the present invention.This type of therapeutical agent includes but not limited to microbiotic, anti-inflammatory agent, matrix metallo-proteinase inhibitor, lipoxidase inhibitor, cytokine antagonist, immunosuppressor, carcinostatic agent, antiviral agent, cytokine, somatomedin, immunomodulator, prostaglandin(PG) or anti-angiogenic hyper-proliferative compound.

Compound of the present invention can be in a usual manner be used to controlling the Endophytic bacteria infection level and being used for the treatment of disease or reducing progress or the seriousness by the effect of bacteria mediated.This type of methods for the treatment of, its dosage level and demand can be selected according to methods availalbe and technology by those of ordinary skills.

For example, compound of the present invention can be with the acceptable adjuvant combination of pharmacy for being applied to the acceptable mode of pharmacy and the amount that effectively alleviates the seriousness of the sort of infection or disease the patient who suffers from bacterium infection or disease.

Alternatively, compound of the present invention can be for composition and method, and it is used for the treatment of or protects individual time period opposing bacterium extending to infect or disease.In one embodiment, compound of the present invention can be for composition and method, and it is used for the treatment of or protects individuality opposing bacterium during 1-2 week to infect or disease.In another embodiment; compound of the present invention can be for composition and method; it is used for the treatment of or protects and individual on opposing bacterium during 4-8 week, infects or disease (for example, suffer from or riskyly develop into endocarditis or myelitic patient in treatment).In another embodiment, compound of the present invention can be for composition and method, and it is used for the treatment of or protects individuality opposing bacterium during 8-12 week to infect or disease.Compound can be used for such composition in the mode consistent with the routine use of enzyme inhibitors in pharmaceutical composition separately or together with other compounds of the present invention.For example, compound of the present invention can be used for routine the acceptable adjuvant combination of pharmacy of vaccine, and uses with the prevention significant quantity, and is individual in time expand section opposing bacterium infection or disease to protect.

In some embodiments, the compound of formula (I) or its pharmacologically acceptable salts can be prophylactically be used to preventing that bacterium from infecting.In some embodiments, the compound of formula (I) or its pharmacologically acceptable salts can be before dentistry or surgical operation, in process or afterwards be used to prevent opportunistic pathogenic infection, those that for example run in bacterial endocarditis.In other embodiments, the compound of formula (I) or its pharmacologically acceptable salts can, prophylactically for dental operation, include but not limited to exodontia, periodontal surgery, dental implant placement and endodontic procedure.in other embodiments, the compound of formula (I) or its pharmacologically acceptable salts can be prophylactically for surgical operations, include but not limited to general surgery, breathe surgery (tonsillectomy/adenoidectomy), gi tract surgery (the little enterochirurgia of top GI and selectivity, esophagus sclerotheraphy and dilatation, large enterectomy, acute appendicectomy), traumatology department's (penetrance abdominal surgery), urogenital tract surgery (prostatectomy, dilation of urethra, cystoscopy, vagina or etroh, cesarean section), spare-part surgery (kidney, liver, pancreas or renal transplantation), (the skin excision of H&N surgery, Neck Dissection, laryngectomy, the head and neck cancer surgery, mandibular fracture), plastic surgery (total joint replacement, the wound open fracture), vascular surgery (peripheral blood vessel operation), cardiothoracic surgery, coronary bypass surgery, pulmonectomy and Neurological Surgery.

As used herein, except as otherwise noted, the antibiosis that term " prevent bacterium from infecting " means gyrase of the present invention for example and/or topoisomerase I V inhibitor usually prevents the preventive use that bacterium infects.Can prophylactically use the treatment of gyrase and/or topoisomerase I V inhibitor to prevent the infection that is caused by the organism to gyrase and/or topoisomerase I V inhibitor sensitivity.One group of general symptom wherein can considering prophylactic treatment be when individual because following reason is easier when infected, for example immunizing power weakens, surgery, wound, Prosthesis having defect in (temporary transient or permanent), dissection, be exposed to high-caliber bacterium and maybe may be exposed to the pathogenic agent that causes disease in vivo.Can cause the example of the factor that immunizing power weakens to comprise that chemotherapy, radiotherapy, diabetes, old age, HIV infect and transplant.In dissection, the example of defect will be valvular defect, and it increases the danger of bacterial endocarditis.Prosthesis's example comprises joint prosthesis, acus, conduit etc.Wherein the prevention of gyrase and/or topoisomerase I V inhibitor use may another suitable group situation can be to prevent the propagation (directly or indirectly) of pathogenic agent between individuality.The specific examples that prevents the preventive use of pathogen propagation is individual gyrase and/or the topoisomerase I V inhibitor of using in health care institution (for example hospital or rest home).

The compound of formula (I) or its pharmacologically acceptable salts can also be used altogether with other microbiotic, to increase for various bacteria treatment of infection or preventive effect.When compound of the present invention during with other agent administration, can be applied to the patient by it in turn or simultaneously in combination therapy.Alternatively, according to medicine of the present invention or prevention composition, comprise the combination of the compound of formula (I) or its pharmacologically acceptable salts and another kind for the treatment of or prevention reagent.

In some embodiments, other one or more therapeutical agents are to be selected from following microbiotic: the penicillin of natural penicillin, penicillinase resistance penicillin, anti-pseudomonas, Aminopenicillin, first generation cephalosporin, second generation cephalosporin, third generation cephalosporin, the 4th generation cynnematin, carbapenem, cephamycin, quinolone, fluoroquinolone, aminoglycoside, macrolide, ketone lactone, polymyxin, tsiklomitsin, glycopeptide, streptogramin, oxazolidone, rifomycin or sulfamido.

In some embodiments, other one or more therapeutical agents are the microbiotic that are selected from penicillin, cynnematin, quinolone, glucosaminide Huo oxazolidone.

in other embodiments, other therapeutical agent is selected from natural penicillin and comprises benzathine penicillin G, penicillin G and penicillin v, be selected from penicillinase resistance penicillin and comprise cloxacillin, dicloxacillin, nafcillin and Oxazacillin, be selected from anti-pseudomonas penicillin and comprise Gepcillin, mezlocillin, piperacillin, piperacillin/Tazobactam Sodium, ticarcillin and ticarcillin/clavulanic acid, be selected from Aminopenicillin and comprise amoxycilline Trihydrate bp, Ampicillin Trihydrate and ampicillin/sulbactam, be selected from first generation cephalosporin and comprise Cephazolin, S 578, Cephalexin Monohydrate Micro/Compacted and Cephradine, be selected from second generation cephalosporin and comprise cefaclor, cefaclor-CD, Cefamandole, cefonicid, Prozef, Loracarbef and cephalofruxin, comprise Cefdinir from third generation cephalosporin, Cefixime Micronized, cefoperazone, cefotaxime, Cefpodoxime, ceftazime, Ceftibuten, ceftizoxime and ceftriaxone, be selected from the 4th generation cynnematin comprise cefepime, CPT (Ceftaroline) and Ceftobiprole, be selected from cephamycin and comprise cefotetan and cefoxitin, is selected from carbapenem and comprises doripenem, imipenum and meropenem, be selected from monobactam (monobactam) and comprise aztreonam, is selected from quinolone and comprises cinoxacin, Nalidixic Acid, oxolinic acid and pipemidic acid, be selected from fluoroquinolone and comprise besifloxacin, Ciprofloxacin, enoxacin, Gatifloxacin, grepafloxacin, levofloxacin, lomefloxacin, Moxifloxacin, norfloxicin, Ofloxacine USP 23 and Sparfloxacin, be selected from aminoglycoside and comprise amikacin, gentamicin, kantlex, Liu Suanyan NEOMYCIN SULPHATE, netilmicin, spectinomycin, Streptomycin sulphate and tobramycin, be selected from macrolide and comprise Azythromycin, clarithromycin and erythromycin, be selected from the ketone lactone and comprise Ketek, is selected from tsiklomitsin and comprises Uromycin, Demethylchlortetracycline, Vibravenos, Minocycline HCl and tsiklomitsin, be selected from glycopeptide and comprise oritavancin, the Da Beiwan star, the Te Lawan star, teicoplanin and vancomycin, be selected from streptogramin and comprise dalfopristin/Quinupristin, and Xuan Zi oxazolidone comprises Linezolid, be selected from rifomycin and comprise rifabutin and Rifampin, and other microbiotic comprises bacitracin, Totazina, Tigecycline, daptomycin, paraxin, clindamycin, vazadrine, metronidazole, mupirocin, PXB, pyrazinoic acid amide, trimethoprim/Sulfamethoxazole and Sulfafurazole.

in other embodiments, other therapeutical agent is selected from natural penicillin and comprises penicillin G, penicillinase resistance penicillin comprises nafcillin and Oxazacillin, anti-pseudomonas penicillin comprises piperacillin/Tazobactam Sodium, Aminopenicillin comprises amoxycilline Trihydrate bp, first generation cephalosporin comprises Cephalexin Monohydrate Micro/Compacted, second generation cephalosporin comprises cefaclor, cefaclor-CD and cephalofruxin, third generation cephalosporin comprises ceftazime and ceftriaxone, the 4th generation cynnematin comprise cefepime, carbapenem comprises imipenum, meropenem, ertapenem, doripenem, panipenem and biapenem, fluoroquinolone comprises Ciprofloxacin, Gatifloxacin, levofloxacin and Moxifloxacin, aminoglycoside comprises tobramycin, macrolide comprises Azythromycin and clarithromycin, tsiklomitsin comprises Vibravenos, glycopeptide comprises vancomycin, rifomycin comprises Rifampin, and other microbiotic comprise vazadrine, pyrazinoic acid amide, Tigecycline, daptomycin or trimethoprim/Sulfamethoxazole.

In some embodiments, the compound of the formula of solid form (I) or its pharmacologically acceptable salts can be applied to the infection for the treatment of Gram-positive.In some embodiments, composition is that for example the form of the compound of formula (I) or its pharmacologically acceptable salts is dissolved into liquid and iv uses for solid, liquid (for example suspension) or iv() composition.in some embodiments, the compound or the composition of its pharmacologically acceptable salts and the other antibiotic agent combined administration that comprise formula (I), described other antibiotic agent is natural penicillin for example, penicillinase resistance penicillin, anti-pseudomonas penicillin, Aminopenicillin, first generation cephalosporin, second generation cephalosporin, third generation cephalosporin, the 4th generation cynnematin, carbapenem, cephamycin, quinolone, fluoroquinolone, aminoglycoside, macrolide, the ketone lactone, polymyxin, tsiklomitsin, glycopeptide, streptogramin, oxazolidone, rifomycin or sulfamido.in some embodiments, comprise that the compound of formula (I) of solid form or the composition oral of its pharmacologically acceptable salts use, and iv. uses for example natural penicillin of other antibiotic agent, penicillinase resistance penicillin, anti-pseudomonas penicillin, Aminopenicillin, first generation cephalosporin, second generation cephalosporin, third generation cephalosporin, the 4th generation cynnematin, carbapenem, cephamycin, quinolone, fluoroquinolone, aminoglycoside, macrolide, the ketone lactone, polymyxin, tsiklomitsin, glycopeptide, streptogramin, oxazolidone, rifomycin or sulfamido.

In some embodiments, the formula of solid form (I) compound or its pharmacologically acceptable salts can be applied to the infection for the treatment of Gram-negative.In some embodiments, composition is that for example the form of the compound of formula (I) or its pharmacologically acceptable salts is dissolved into liquid and iv uses for solid, liquid (for example suspension) or iv() composition.In some embodiments, the composition that comprises the compound of formula (I) or its pharmacologically acceptable salts be selected from following other antibiotic agent combined administration: natural penicillin, penicillinase resistance penicillin, anti-pseudomonas penicillin, Aminopenicillin, first generation cephalosporin, second generation cephalosporin, third generation cephalosporin, the 4th generation cynnematin, carbapenem, cephamycin, monobactam, quinolone, fluoroquinolone, aminoglycoside, macrolide, ketone lactone, polymyxin, tsiklomitsin or sulfamido.In some embodiments, comprise that the compound of formula (I) of solid form or the composition oral of its pharmacologically acceptable salts use, and Orally administered other antibiotic agent for example natural penicillin, penicillinase resistance penicillin, anti-pseudomonas penicillin, Aminopenicillin, first generation cephalosporin, second generation cephalosporin, third generation cephalosporin, the 4th generation cynnematin, carbapenem, cephamycin, monobactam, quinolone, fluoroquinolone, aminoglycoside, macrolide, ketone lactone, polymyxin, tsiklomitsin or sulfanilamide (SN).In some embodiments, iv uses other therapeutical agent.

Above-described other therapeutical agent can be used as the part of multiple doses scheme and uses respectively with the composition that contains inhibitor.Alternatively, these reagent can be that the part of one-pack type is mixed together in single composition with inhibitor.

Pharmaceutical composition of the present invention can be oral, parenteral, by suction spray, outside, rectum, nose, cheek, vagina or use via the embedded type holder.Pharmaceutical composition of the present invention can contain nontoxic pharmaceutically acceptable carrier, adjuvant or the vehicle of any routine.In some cases, the pH of preparation can use the acceptable acid of pharmacy, alkali or damping fluid adjustment, with the compound of enhancing preparation or the stability of its delivery form.As used herein term parenterally comprise in subcutaneous, intracutaneous, intravenously, intramuscular, intraarticular, synovial membrane, in breastbone, in sheath, intralesional and intracranial injection or infusion techniques.

Pharmaceutical composition can be with the form of sterile injectable preparation, for example as sterile injectable water-based or oily suspensions.This suspension can be prepared according to technology known in the art, uses suitable dispersion agent or wetting agent (for example Tween80) and suspension agent.Sterile injectable preparation can also be sterile injectable solution or the suspension in the acceptable thinner of nontoxic parenteral or solvent, for example solution in 1,3 butylene glycol.Adoptable in acceptable vehicle and solvent is N.F,USP MANNITOL, water, Ringer's solution and isotonic sodium chlorrde solution.In addition, adopt routinely aseptic fixed oil as solvent or suspension medium.For this purpose, the fixed oil of any gentleness be can adopt, synthetic glycerine monoesters or triglyceride comprised.Lipid acid for example oleic acid and glyceride derivative thereof is useful in the injection preparation, and similarly the acceptable oil of natural pharmacy is also useful, and for example sweet oil or Viscotrol C, especially with its polyoxyethylene form.These oil solutions or suspension also can contain long-chain alcohol thinner or dispersion agent, those that for example describe in Pharmacopeia Helvetica, or similar oral alcohol.

Pharmaceutical composition of the present invention can come with any oral acceptable formulation Orally administered, includes but not limited to capsule, tablet and aqeous suspension and solution.In the situation that the tablet be used to orally using, normally used carrier comprises lactose and W-Gum.Usually also add for example Magnesium Stearate of lubricant.Orally administered for capsule form, useful thinner comprises lactose and dried corn starch.When aqeous suspension and solution and polyoxyethylene glycol when Orally administered, activeconstituents and emulsifying agent and suspension agent combination.While needing, can add certain sweeting agent and/or seasonings and/or tinting material.

Pharmaceutical composition of the present invention can also be used with the suppository form for rectal administration.These compositions can be by compound of the present invention is mixed and is prepared with suitable non-irritating excipient, described vehicle room temperature be solid but under rectal temperature, be liquid, and therefore will in rectum, melt with the release active ingredient.Materials includes but not limited to theobroma oil, beeswax and polyoxyethylene glycol.

When but required treatment related to the applications accessible zone of appearance or organ, the external application of pharmaceutical composition of the present invention was particularly useful.For the applications to skin, pharmaceutical composition should be prepared with suitable ointment, and described ointment contains suspension or is dissolved in the active ingredient in carrier.The carrier that is used for the external application of compound of the present invention includes but not limited to mineral oil, liquid petroleum, white oil, propylene glycol, polyoxyethylene, polyoxypropylene, emulsifying wax and water.Alternatively, pharmaceutical composition can be prepared with suitable emulsion or creme, and described emulsion or creme contain suspension or be dissolved in the active ingredient in carrier.Suitable carrier includes but not limited to mineral oil, Sorbitol Powder monostearate, polysorbate60, cetyl esters wax, cetostearyl alcohol, 2-Standamul G, phenylcarbinol and water.Pharmaceutical composition of the present invention can also be applied to lower intestine by the rectal suppository preparation or at suitable enema preparation peripheral.The transdermal patch of external application is also included within the present invention.

Pharmaceutical composition of the present invention can be used by aerosol or the inhalation of nose.Such composition is prepared according to the well-known technology of field of pharmaceutical preparations, and can be prepared into the solution in physiological saline, it adopts phenylcarbinol or other suitable sanitass, absorption enhancer to strengthen bioavailability, fluorocarbon and/or other stablizers known in the art or dispersion agent.

According to another embodiment, the compound of formula (I) or its pharmacologically acceptable salts also can for example be sent with implantable or indwelling equipment by implanting (for example surgery).Implantable or indwelling equipment can be designed as forever or temporarily and resides in the experimenter.Implantable and example indwelling equipment includes but not limited to contact lenses, central venous catheter and Needleless connector, endotracheal intubation, intrauterine device, mechanical heart valve, pacemaker, peritoneal dialysis catheters, pseudarthrosis for example hip and knee replacement, TT, urinary catheter, Voice prosthesis, support, transferpump, vascular filter and implantable control release composition.Biofilm can be harmful to the patient health with implantable or indwelling equipment, because they are introduced artificial substratum in body and can cause persistent infection.Therefore, among implantable or indwelling equipment or above the compound of formula (I) or the generation that its pharmacologically acceptable salts could prevent or reduce biofilm are provided.What in addition, implantable or indwelling equipment can be used as the compound of formula (I) or its pharmacologically acceptable salts accumulates place or holder.Any implantable or indwelling equipment may be used to compound or its pharmacologically acceptable salts of delivery type (I), condition is compound or its pharmacologically acceptable salts of a) described device, formula (I) and comprises that the compound of formula (I) or any pharmaceutical composition of its pharmacologically acceptable salts are biocompatible, and b) compound or its pharmacologically acceptable salts of the formula (I) of significant quantity can be sent or discharge to described device, with the patient to treatment, gives curative effect.

Therapeutical agent is known in the art by sending of implantable or indwelling equipment.Referring to for example, by people such as Hofma at Current Interventional Cardiology Reports2001, " the Recent Developments in Coated Stents " that delivers in 3:28-36, its complete content comprise that the reference of wherein quoting integrates with this paper by reference.Other descriptions of implantable device can be at U.S. Patent number 6,569, and 195 and 6,322,847; And find in U.S. Patent Application No. 2004/0044405,2004/0018228,2003/0229390,2003/0225450,2003/0216699 and 2003/0204168, each described patent integral body is by reference integrated with this paper.

In some embodiments, implantable device is support.In a particular, support can comprise the netted cable of interlocking.Every cable can comprise for the metal wire of structural support with for the polymerization line of delivering therapeutic agents.The polymerization line can carry out administration by polymkeric substance being immersed in treatment agent solution.Alternatively, in the process that therapeutical agent can be online formed by the polymerization precursor solution, be embedded in the polymerization line.

In other embodiments, implantable or indwelling equipment can be coated with the polymeric coatings that comprises therapeutical agent.Polymeric coatings can be designed as the rate of release of controlling therapeutical agent.The control of therapeutical agent discharges can utilize multiple technologies.Known that device has whole layer or dressing, it is incorporated into the heterogeneous solution of promoting agent and/or dispersion in polymeric material, wherein the diffusion of reagent is rate limiting, because reagent arrives polymkeric substance-liquid surface by polymer diffusion, and is discharged in surrounding liquid.In some devices, soluble material also dissolves or is dispersed in polymeric material, makes and after material dissolves, stays other hole or passage.Matrix device is also generally diffusional limitation, but passage or other interior geometry of device also are discharged in liquid and work at reagent.Passage can also be the passage that is pre-existing in or by the passage that stays after release reagent or other soluble materials.

Erodable or degradable device physically are immobilized in promoting agent in polymkeric substance usually.Promoting agent can dissolve and/or disperse to spread all over polymeric material.Polymeric material is degraded with hydrolytic action in time by the hydrolysis of labile bond usually, allows polymer erodes in liquid, and promoting agent is discharged in liquid.Hydrophilic polymer has usually erosion ratio faster with respect to hydrophobic polymer.Hydrophobic polymer is considered to have the almost pure surface diffusion of promoting agent, has the erosion inside from surface.Hydrophilic polymer is considered to allow the surface of penetration by water polymkeric substance, allows in subsurface labile bond hydrolysis, and this can cause uniform or a large amount of erosion of polymkeric substance.

Dressing implantable or indwelling equipment can comprise the polymkeric substance admixture, and it has different therapeutical agent release rates separately.For example, dressing can comprise poly(lactic acid)/polyoxyethylene (PLA-PEO) multipolymer or poly(lactic acid)/polycaprolactone (PLA-PCL) multipolymer.With respect to poly(lactic acid)/polycaprolactone (PLA-PCL) multipolymer, poly(lactic acid)/polyoxyethylene (PLA-PEO) multipolymer can demonstrate higher therapeutical agent release rate.The relative quantity of the therapeutical agent of sending in time and the speed of dosage can be controlled for the relative quantity of the polymkeric substance that more slowly discharges by controlling the polymer phase that discharges more fast.For higher initial release rate, the ratio of the polymkeric substance that discharges more fast can increase with respect to the polymkeric substance that more slowly discharges.If most of dosage need to discharge through long-time section, most polymers can be the polymkeric substance that more slowly discharges so.The device dressing can be sprayed to device by the solution with polymkeric substance, promoting agent and solvent or dispersion.Solvent can evaporate, and stays the dressing of polymkeric substance and promoting agent.Promoting agent can dissolve and/or be dispersed in polymkeric substance.In some embodiments, multipolymer can be extruded on device.

The about 100mg/kg body weight/day of about 0.01-, the preferred about 75mg/kg body weight/day of 0.5-and most preferably from about the dosage level of the active compound component of 1-50mg/kg body weight/day for preventing and treat that the monotherapy that bacterium infects is useful.

Usually, pharmaceutical composition of the present invention will be used approximately 1 – 5 times every day or alternatively as continuous infusion.Alternatively, composition of the present invention can be used in the pulse preparation.This type of is used and can be used as chronic or acute treatment.The amount that can make up to produce the activeconstituents of single formulation with solid support material changes the host of Dependence Treatment and concrete mode of administration.Exemplary formulations will contain approximately 95% the active compound (w/w) of 5%-of having an appointment.Preferably, this type of preparation contains approximately 80% active compound of 20%-of having an appointment.

When composition of the present invention comprised the combination of the compound of formula (I) or its pharmacologically acceptable salts and one or more other therapeutical agents or preventive, described compound and other reagent should exist with the dosage level of the approximately 10%-80% dosage usually used in the monotherapy scheme.

After patient's symptom is improved, while needing, can use compound of the present invention, composition or the combination of maintenance dose.Subsequently, according to symptom, the dosage of using or frequency or both whiles, as the function of symptom, can be reduced to the level of the symptom that maintains improvement, when symptom had alleviated to desired level, treatment should stop.Yet, based on any recurrence or disease symptoms patient may be on long-term basis the gap therapy.

As should be understood as technical staff, may need than those lower or higher dosage mentioned above.For any concrete patient's given dose and treatment plan, will rely on many factors, comprise that the seriousness of activity, age, body weight, general health state, sex, diet, time of application, excretion rate, drug regimen, disease of the specific compound of employing and process and patient are to the psychological tendency of disease with to treating doctor's judgement.

According to another embodiment, the invention provides and be used for the treatment of or prevent that bacterium from infecting or the method for morbid state, it comprises the step of using any compound described herein, pharmaceutical composition or combination to the patient.As used herein, term " patient " means animal, preferred mammal, and be most preferably the people.

Compound of the present invention is also as commercial reagents, itself and gyrase B and/or the effective combination of topoisomerase I V enzyme.As commercial reagents, compound of the present invention and derivative thereof can, at the biological chemistry for bacteria gyrase B and/or topoisomerase I V or its homologue or raji cell assay Raji be used to blocking the activity of gyrase B and/or topoisomerase I V, maybe can derive as with stable resin, being combined and apply for affinity chromatography as tying substrate.These and other purposes that characterize business gyrase B and/or topoisomerase I V inhibitor will be apparent for those of ordinary skills.

In order to understand more comprehensively the present invention, set forth following proposal and embodiment.These embodiment are only presented for purposes of illustration, and should not be construed as by any way and limit the scope of the invention.

Following definitions is described term as herein described and abbreviation:

The Ac ethanoyl

The Bu butyl

The Et ethyl

The Ph phenyl

The Me methyl

The THF tetrahydrofuran (THF)

The DCM methylene dichloride

CH ₂Cl ₂Methylene dichloride

The EtOAc ethyl acetate

CH ₃The CN acetonitrile

EtOH ethanol

Et ₂The O diethyl ether

MeOH methyl alcohol

The MTBE methyl tert-butyl ether

The DMF DMF

The DMA N,N-dimethylacetamide

The DMSO methyl-sulphoxide

HOAc acetic acid

The TEA triethylamine

The TFA trifluoroacetic acid

The TFAA trifluoroacetic anhydride

Et ₃The N triethylamine

The DIPEA diisopropylethylamine

The DIEA diisopropylethylamine

K ₂CO ₃Salt of wormwood

Na ₂CO ₃Sodium carbonate

Na ₂S ₂O ₃Sulfothiorine

Cs ₂CO ₃Cesium carbonate

NaHCO ₃Sodium bicarbonate

NaOH sodium hydroxide

Na ₂SO ₄Sodium sulfate

MgSO ₄, sal epsom

K ₃PO ₄Potassiumphosphate

NH ₄Cl ammonium chloride

LC/MS liquid phase chromatography/mass spectrum

GCMS vapor-phase chromatography mass spectrum

The HPLC high performance liquid chromatography

The GC vapor-phase chromatography

The LC liquid phase chromatography

The IC ion chromatography

The IM intramuscular

The CFU/cfu colony-forming unit

The MIC minimum inhibitory concentration

Hr or h hour

The atm normal atmosphere

Rt or RT room temperature

The TLC tlc

HCl hydrochloric acid

H ₂O water

The EtNCO ethyl isocyanate

Pd/C palladium carbon

The NaOAc sodium acetate

H ₂SO ₄Sulfuric acid

N ₂Nitrogen

H ₂Hydrogen

The n-BuLi n-Butyl Lithium

DI is deionized

Pd(OAc) ₂Acid chloride (II)

PPh ₃Triphenylphosphine

The i-PrOH Virahol

The NBS N-bromosuccinimide

Pd[(Ph ₃) P] ₄Tetrakis triphenylphosphine palladium (0)

The PTFE tetrafluoroethylene

The rpm rotations per minute

The SM starting material

Equiv. equivalent

¹The H-NMR proton magnetic resonance (PMR)

Synthesizing of compound

Embodiment

6-fluorobenzene imidazolyl carbamide compound

(R) the fluoro-5-(2-(2-oxyacetone of-1-ethyl-3-(6--2-yl) pyrimidine-5-yl)-7-(tetrahydrofuran (THF)-2-yl)-1 hydrogen-benzimidazolyl-2 radicals-yl) urea is synthetic

Scheme 3 provides the method for the preparation of 6-fluorobenzene imidazolyl carbamide compound.

Scheme 3

Embodiment 1.a

The fluoro-6-nitro-phenyl of 2-(2-)-2,3 dihydro furan (15A) and 2-(2-fluorine 6-nitro-phenyl)-preparation of DHF (15B)

By the fluoro-3-nitro-benzene of the bromo-1-of 2-(14) (200.3g, 98%, 892.3mmol, Bosche F6657), 1,4-diox (981.5mL, Sigma-Aldrich360481) and 2,3 dihydro furan (2) (341.1mL, 99%, 4.462mol, Aldrich200018) pack in reaction vessel, add subsequently DIPEA (155.4mL, 892.3mmol, Sigma-Aldrich550043) and bromine (tri-butyl phosphine) palladium (I) binary (6.936g, 8.923mmol, Johnson Matthey C4099).Reaction mixture is stirred to 2 hours (consumption of the initial aryl bromide of HPLC demonstration 98%) under refluxing.The permission reaction mixture is cooling, with filtering, shifts out precipitation, with EtOAc, rinses, and filtrate is condensed into to the dun lard under vacuum.Described lard is dissolved in methylene dichloride, and with methylene dichloride process silicon-dioxide embolism wash-out, and concentrated the generation is the 15A of dark amber oil and the mixture of 15B (291.3g) under vacuum.This crude product advances without being further purified.Main products is the fluoro-6-nitro-phenyl of 2-(2-)-2,3 dihydro furan (15A) (96%): LCMS (uses 10-90%CH ₃CN/ water gradient elution C18 post 5 minutes, use formic acid properties-correcting agent) M+1:210.23 (3.13 minutes); ¹H NMR (300MHz, CDCl ₃) δ 7.54 (dt, J=8.0,1.2Hz, 1H), 7.43 (td, J=8.2,5.2Hz, 1H), 7.32 (ddd, J=9.7,8.3,1.3Hz, 1H), 6.33 (dd, J=4.9,2.4Hz, 1H), 5.80 (t, J=10.9Hz, 1H), 5.06 (q, J=2.4Hz, 1H), 3.18 – 3.07 (m, 1H), 2.94 – 2.82 (m, 1H) ppm.The minority product is 2-(2-fluorine 6-nitro-phenyl)-DHF (15B) (4%): GCMS (Agilent HP-5MS30m x250 μ m x0.25 μ m pillar 60 ℃ of heating reached in 2 minutes 300 ℃ with 1mL/ minute flow velocity through 15 minutes) M+1:210 (11.95 minutes). ¹H?NMR(300MHz,CDCl ₃)δ7.47(d,J=8.0Hz,1H),7.43–7.34(m,1H),7.30–7.23(m,1H),6.21–6.15(m,1H),6.11–6.06(m,1H),5.97–5.91(m,1H),4.89–4.73(m,2H)ppm.

Embodiment 1.b

The preparation of the fluoro-2-tetrahydrofuran (THF) of 3--2-base-aniline (16)

5% carbon palladium (37.3g, 50% is moistening, 8.76mmol, Aldrich330116) is placed in to the Parr bottle under nitrogen, adds subsequently MeOH(70mL, JT-Baker909333).To be dissolved in MeOH(117mL) in the fluoro-6-nitro-phenyl of 2-(2-)-2,3 dihydro furan and 2-(2-fluorine 6-nitro-phenyl)-DHF (15A& 15B) crude mixture of (186.6g, 892.1mmol) adds in the Parr bottle, adds subsequently NEt ₃(124.3mL, 892.1mmol, Sigma-Aldrich471283).Be placed in bottle on the Parr vibrator and use H ₂Saturated.Add 45psi H ₂Afterwards, the oscillatory reaction mixture is until starting material completely consumed (HPLC and LCMS show complete reaction).Reaction mixture is purified with nitrogen, pass through Celite ^TMFilter and rinse with EtOAc.Filtrate is condensed into to brown oil on rotatory evaporator, it is dissolved in Et ₂O also washes (2x) with water.Ether is used water-soluble 1N HCl (5x250mL) extraction mutually, uses Et ₂O washes (3x) and then with water-soluble 6NNaOH, alkalizes to pH12-14.Alkalescence water dichloromethane extraction (CH ₂Cl ₂, 4x), and the organic extract after merging is with saturated water-soluble NH ₄Cl, at MgSO ₄Upper drying, and filter and use CH by the silicon-dioxide bed course ₂Cl ₂Be eluted to the 25%EtOAc/ hexane.Required filtrate is concentrated 16 (121.8g, 84%GCMS add NMR purity) that produce as shallow brown oil under reduced pressure.GCMS (Agilent HP-5MS30m x250 μ m x0.25 μ m pillar 60 ℃ of heating reached in 2 minutes 300 ℃ with 1mL/ minute flow velocity through 15 minutes) M+1:182.0 (11.44 minutes).LCMS (uses 10-90%CH ₃CN/ water gradient elution C18 post 5 minutes, use formic acid properties-correcting agent) M+1:182.10 (2.61 minutes). ¹H NMR (300MHz, CDCl ₃) δ 6.97 (td, J=8.1,6.3Hz, 1H), 6.43 – 6.35 (m, 2H), 5.21 – 5.13 (m, 1H), 4.54 (s, 2H), 4.16 – 4.07 (m, 1H), 3.90 – 3.81 (m, 1H), 2.23 – 2.00 (m, 4H) ppm.The following acquisition of a collection of harvest in addition: the ether after merging is used saturated water-soluble NaHCO mutually ₃, bittern washes, at Na ₂SO ₄Upper drying, decantation under reduced pressure concentrated.Described oil is collected distillment through vacuum distilling (ca.15torr) 108 ℃ of 101 –.5M HCl (1eq) in 2 ℃ will be dissolved in iPrOH slowly joins and distills to obtain oily stirred solution in EtOH (1 part of volume).Gained suspension is placed in room temperature, and (3 parts of volumes vol/vol) dilute and stirred 2 hours with EtOAc.Filter and collect white solid, with EtOAc, wash also drying under reduced pressure and produce the second batch harvest as hydrochloride.Maternal liquid concentration form slurry, with EtOAc dilution solid collected by filtration, produce the three batch harvest of hydrochloride as described product with EtOAc washing vacuum-drying.LCMS(10-90%CH ₃CN/ water gradient elution C18 post 5 minutes, use formic acid properties-correcting agent) M+1:182.10(2.58 minute). ¹H?NMR（300MHz，CDCl ₃）δ10.73(br.s,3H),7.66(d,J=8.1Hz,1H),7.33(td,J=8.2,5.9Hz,1H),7.13–7.05(m,1H),5.26(dd,J=9.0,6.5Hz,1H),4.38–4.28(m,1H),4.00–3.91(m,1H),2.59–2.46(m,1H),2.30–1.95(m,3H)ppm。Overall yield from three batches of harvests is 76%.

Embodiment 1.c

The preparation of the fluoro-2-tetrahydrofuran (THF) of the bromo-3-of 4--2-base-aniline (17)

To the fluoro-2-tetrahydrofuran (THF) of the 3-that is cooled to-20 ℃-2-base-aniline (16) (131.9g, 92%, 669.7mmol) add 3 parts of N-bromine succinimide (120.4g in stirred solution in methyl tert-butyl ether (1.456L) and acetonitrile (485mL), 99%, 669.7mmol, Aldrich B81255), remain temperature of reaction lower than approximately-15 ℃.After interpolation completes, continue to stir 30 minutes at-15 to-10 ℃.The work aliquots containig ¹H NMR shows raw materials consumption 96%.Other 4.82gNBS joins reaction mixture, at-10 ℃, stirs extra 30 minutes simultaneously.By water-soluble 1N Na ₂S ₂O ₃(670mL) add in reaction mixture.Remove cooling bath and stirred the mixture 20 minutes, with EtOAc, diluting.Layering.By organic phase with saturated water-soluble NaHCO ₃(2x), the washing of water, bittern, at Na ₂SO ₄Upper drying, decantation and under reduced pressure concentratedly obtain dark amber oil.Resistates dilutes with hexane and uses the 25%-50%EtOAc/ hexane by silicon-dioxide embolism wash-out.Required filtrate is concentrated in a vacuum, produces 17 of dark amber oil EtOAc/ hexane.LCMS(uses 10-90%AcN/ water gradient elution C18 post 5 minutes, uses formic acid properties-correcting agent) M+1:260.12 (3.20min). ¹H?NMR(300MHz,CDCl ₃)δ7.15(dd,J=8.6,7.6Hz,1H),6.30(dd,J=8.7,1.3Hz,1H),5.19–5.12(m,1H),4.58(s,2H),4.16–4.07(m,1H),3.90–3.81(m,1H),2.23–1.99(m,4H)ppm。

Embodiment 1.d

The fluoro-6-nitro of the bromo-3-of N-(4--2-tetrahydrofuran (THF)-2-base-phenyl)-2,2, the preparation of 2-trifluoroacetamide (18)

Will be as the fluoro-2-tetrahydrofuran (THF) of the bromo-3-of anhydrous (neat) 4-of toughening oil-2-base-aniline (17) (123.0g, 86%, 406.7mmol) by adding funnel with the trifluoroacetic anhydride (565.3mL that slowly joined 2 ℃ of stirrings in 20 minutes, 4.067mol, Sigma-Aldrich106232) (temperature of reaction rises to 13 ℃).By the anhydrous THF(35mL of remaining oil) be flushed in reaction mixture.Remove cooling bath and reactant and be heated to 35 ℃, with 2.5 hours, by part interpolation ammonium nitrate (4.88gX20 part, 1.22mol, Sigma-Aldrich A7455), keep temperature of reaction at about 30-41 ℃, only with cooling bath, control heat release when needed.After interpolation completed, reaction mixture stirred other 10 minutes (the HPLC reaction completes 99%) again.By the slow impouring reaction mixture stir and allowed filtrable solid sediment to form in 1 hour of trash ice (1.23kg), collect described solid sediment and wash, use briefly saturated water-soluble NaHCO with water ₃Again wash (to pH7) with water.Product in normal reheating furnace 40 ℃ of dried overnight, then under reduced pressure in process furnace 50 ℃ of dried overnight obtain being the 18(152.5g of filbert solid, 90% productive rate; 96%HPLC purity).LCMS(10-90%CH ₃CN/ water gradient elution C18 post 5 minutes, use formic acid properties-correcting agent) M+1:401.30 (3.41 minutes). ¹H NMR (300MHz, CDCl ₃) δ 10.56 (s, 1H), 8.19 (d, J=6.6Hz, 1H), 5.22 (dd, J=10.3,6.4Hz, 1H), 4.22 (dd, J=15.8,7.2Hz, 1H), 3.99 (dd, J=16.1,7.5Hz, 1H), 2.50 – 2.38 (m, 1H), 2.22 – 2.11 (m, 2H), 1.86 – 1.71 (m, 1H) ppm.

Embodiment 1.e

The preparation of the fluoro-6-nitro of the bromo-3-of 4--2-tetrahydrofuran (THF)-2-base-aniline (19)

By the fluoro-6-nitro of the bromo-3-of N-(4--2-tetrahydrofuran (THF)-2-base-phenyl)-2,2,2-trifluoroacetamide (18) (242.3g, 604.1mmol), 1,4-diox (1.212L) and water-soluble 2M sulfuric acid (362.4mL, 724.9mmol) install in reaction flask, and under refluxing, stir 5 days (HPLC shows that complete 98% transforms).The permission mixture is cooling, with the EtOAc dilution, with saturated water-soluble NaHCO ₃Neutralization, layering, and with EtOAc aqueous phase extracted (2X) again.The organic phase that merges is washed (2X) with bittern, at MgSO ₄Upper drying, filter and vacuum concentration produces the 19(181.7g that is dark-coloured succinol, 94% productive rate; Overall 95%HPLC purity).Product is advanced to next step without being further purified.LCMS(10-90%CH ₃CN/ water gradient elution C18 post 5 minutes, use formic acid properties-correcting agent) M+1:305.20 (3.63min) .1H NMR (300MHz, CDCl ₃) δ 8.35 (d, J=7.3Hz, 1H), 7.45 (s, 2H), 5.23 – 5.16 (m, 1H), 4.23 – 4.14 (m, 1H), 3.93 – 3.84 (m, 1H), 2.31 – 1.96 (m, 4H) ppm.

Embodiment 1.f

The fluoro-5-nitro of 2-[5-(4-amino-2--3-tetrahydrofuran (THF)-2-base-phenyl) pyrimidine-2-base] preparation of propan-2-ol (20).

To 1,4-diox (4.20L, in the stirred solution of the fluoro-6-nitro of the bromo-3-of the 4-Sigma-Aldrich360481)-2-tetrahydrofuran (THF)-2-base-aniline (19) (525.0g, 1.721mol, Bridge Organics Co.), add 1.2M aqueous solution NaHCO ₃(4.302L, 5.163mol).Nitrogen is flowed to bubbling by stirring the mixture 2 hours, add subsequently 2-[5-(4,4,5,5-tetramethyl--1,3,2-dioxy boron, penta ring-2-yl) pyrimidine-2-base] the third-2 alcohol (7) (545.4g, 2.065mol, Bridge Organics Co.) and 1,1 '-bis-(diphenylphosphine) ferrocene dichloro palladium methylene dichloride adducts (42.16g, 51.63mmol, Strem460450).Reaction mixture is stirred and spends the night under refluxing, cooling, use EtOAc(8.4L) and dilution, layering.Organic phase is with saturated water-soluble NH ₄Cl and bittern washing.Use EtOAc(4L) aqueous phase extracted wash organic phase with bittern again.The organic phase that merges is at MgSO ₄Upper drying, and pass through

Short plug filter, use the EtOAc wash-out, filtrate is the moistening solid of concentrated generation dark-brown on rotatory evaporator.This solid is dissolved in CH ₂Cl ₂In, be added on the silicon-dioxide rubber cushion, use the hexane wash-out, 25%EtOAc/ hexane and 50%EtOAc/ hexane wash-out subsequently subsequently.Required cut is concentrated to form dense thick suspension on rotatory evaporator, and solid collected by filtration, grind with MTBE, and drying produces the 20(55.8% productive rate as the glassy yellow solid, 90-97%HPLC purity under vacuum).Concentrated filtrate also repeats above-mentioned purifying and produces 20 the second batch harvest (19.7% productive rate) that is the glassy yellow solid.Concentrated filtrate produces dark-brown oil and it is added to silicon-dioxide pillar toluene and minimum CH again ₂Cl ₂.Above-mentioned with EtOAc/ hexane (0%-50%) wash-out.Required cut is condensed into slurries and dilutes with the MTBE/ hexane.Solid collected by filtration, produce with minimum MTBE washing the 20(4.9% productive rate of the 3rd batch that is the glassy yellow solid), its overall yield from three batches of harvests is 80%.LCMS(10-90%CH ₃CN/ water gradient elution C18 post 5 minutes, use formic acid properties-correcting agent) M+1:363.48 (2.95 minutes). ¹H?NMR(300MHz,CDCl ₃)δ8.84(d,J=1.6Hz,2H),8.27(d,J=8.0Hz,1H),7.62(s,2H),5.31–5.24(m,1H),4.63(s,1H),4.27–4.18(m,1H),3.97–3.87(m,1H),2.33–2.05(m,4H),1.64(s,6H)ppm。

Embodiment 1.g

2-[5-(4, the fluoro-3-tetrahydrofuran (THF) of 5-diamino-2--2-base-phenyl) pyrimidine-2-base] preparation of propan-2-ol (21).

Under nitrogen, in the Parr bottle, put into 5% carbon palladium (14.21g, 50%wet, 3.339mmol, Aldrich330116), then add MeOH(242mL, JT-Baker909333) and NEt ₃(46.54mL, 333.9mmol, Sigma-Aldrich471283).The fluoro-5-nitro of 2-[5-(4-amino-2--3-tetrahydrofuran (THF)-2-base-phenyl) pyrimidine-2-base] propan-2-ol (20) (121.0g, 333.9mmol) be dissolved in heat THF(360mL), allow cooling, join in above-mentioned reaction mixture, and with another part THF(124mL) the washing residual volume 20.Be placed in reaction flask on the Parr vibrator and use H ₂Saturated.Adding 45psi H ₂After, reaction flask is vibrated until 20 consumption complete (HPLC and LCMS show complete reaction).Reaction mixture is purified with nitrogen, pass through Celite ^TMFilter and rinse with EtOAc.Filtrate is refiltered by filter paper (glass fibre), and vacuum concentrated filtrate.With identical scale, repeat above-mentioned reaction 3 times again, merge the 21(447g that batch generation is brown solid, 99% productive rate; 93%HPLC purity).LCMS(10-90%CH ₃CN/ water gradient elution C18 post 5 minutes, use formic acid properties-correcting agent) M+1:333.46 (1.79min). ¹H NMR (300MHz, CDCl ₃) δ 8.81 (d, J=1.4Hz, 2H), 6.69 (d, J=7.3Hz, 1H), 5.27 – 5.20 (m, 1H), (4.73 s, 1H), 4.70 (s, 2H), 4.23 – 4.14 (m, 1H), 3.94 – 3.86 (m, 1H), (3.22 s, 2H), 2.32 – 2.22 (m, 1H), 2.18 – 1.99 (m, 3H), 1.63 (s, 6H) ppm.

Embodiment 1.h

The fluoro-5-[2-(1-hydroxyl of 1-ethyl-3-[6--1-methyl-ethyl) pyrimidine-5-yl]-7-tetrahydrofuran (THF)-2-base-1 hydrogen-benzimidazolyl-2 radicals-yl] preparation of urea (22).

to 2-[5-(4, the fluoro-3-tetrahydrofuran (THF) of 5-diamino-2--2-base-phenyl) pyrimidine-2-base] propan-2-ol (21) (111.3g, 334.9mmol) and 1, 4-diox (556.5mL, Sigma-Aldrich360481) in the stirred suspension in, add 1-ethyl-3-(N-(ethyl carbamyl)-C-methyl sulfo--carbon imino-) urea (10) (93.36g, 401.9mmol, CB Research and Development), add subsequently pH3.5 damping fluid (1.113L), described pH3.5 damping fluid is by being dissolved in the water-soluble H of 1N by NaOAc trihydrate (158.1g) ₂SO ₄(1.100L) preparation in.Reaction mixture stirs spend the night (HPLC shows conversion fully) under refluxing, cool to room temperature, and be poured into water-soluble saturated NaHCO by part (reduce and bubble to greatest extent) ₃(2.23L), provide pH8-9.The mixture that produces was stirred 30 minutes, and by solid collected by filtration, water fully washs to neutral pH, and subsequently with the washing of EtOH less amount ground.The solid drying under reduced pressure, be (22) (135.2g, 94% productive rate of yellowy white solid; 99%HPLC purity).LCMS(10-90%CH ₃CN/ water gradient elution C18 post 5 minutes, use formic acid properties-correcting agent) M+1:429.58 (2.03min). ¹H?NMR(300MHz,MeOD)δ8.95(d,J=1.6Hz,2H),7.45(d,J=6.5Hz,1H),5.38(br.s,1H),4.27(dd,J=14.9,7.1Hz,1H),4.01(dd,J=15.1,7.0Hz,1H),3.37–3.29(m,2H),2.55(br.s,1H),2.19–2.07(m,2H),2.02–1.82(br.s,1H),1.63(s,6H),1.21(t,J=7.2Hz,3H)ppm。

Embodiment 1.i

The fluoro-5-[2-(1-hydroxyl of 1-ethyl-3-[6--1-methyl-ethyl) pyrimidine-5-yl]-7-[(2R)-tetrahydrofuran (THF)-2-yl]-1 hydrogen-benzimidazolyl-2 radicals-yl] chiral chromatography of urea (23) separates

(by Chiral Technologies) resolves the fluoro-5-[2-(1-hydroxyl of (resolve) 1-ethyl-3-[6--1-methyl-ethyl) pyrimidine-5-yl]-7-tetrahydrofuran (THF)-2-base-1H-benzimidazolyl-2 radicals-yl] urea (22) racemize sample (133.60g), at 25 ℃, using CH ₂Cl ₂/ MeOH/TEA(60/40/0.1) wash-out, provide the required enantiomorph (23) (66.8g, 45% yield that are the off-white color solid; 99.8%HPLC purity, 99+%ee).Analytical chirality HPLC retention time be 7.7 minutes ( 4.6x250mm post, 1mL/ minute flow velocity, 30 ℃).Described solid suspension is in 2:1EtOH/Et ₂O(5 times of volume), stirred 10 minutes, filter and collect, use 2:1EtOH/Et ₂O washes and drying under reduced pressure produces white solid (60.6g).

By the single-crystal x x ray diffraction analysis x, confirm 23 structure and absolute stereo chemistry.In outfit sealed tube Cu K-α source (Cu K α radiation,

) and the Bruker Apex II diffractometer of Apex II CCD detector on obtain the single crystal diffraction data.Selection has the crystal of 0.15x0.15x0.10mm size, uses mineral oil clean, on MicroMount, fixes and be placed in the center of Bruker APEXII system.Acquisition separates in reciprocal space three batches of 40 frames, to provide direction matrix and initial unit cell parameters.After completing, data gathering obtains and the final unit cell parameters of refining based on whole set of data.Based on systematic absence and intensity statistics, and at acentric P2 ₁In spacer, resolve and its structure of refining.

Use is used 0.5 ° of step-length for the exposure of 30 seconds of every frame, obtains to arrive

The diffraction data collection of reciprocal space of resolving power.At 100(2) collect data during K.Use APEXII software to complete the refining of intensity integration and lattice parameter.The observation to crystal after data gathering shows without decomposing sign.As shown in Figure 2, in structure, having two symmetrical independent molecules and two symmetrical independent molecules is all the R isomer.

Use the collection of Apex II software, refining and reduced data.Use SHELXS97(Sheldrick, 1990) the program analytic structure, and use SHELXL97(Sheldrick, 1997) program refining structure.Crystal shows to have P2 ₁The monocline structure cell of spacer.Lattice parameter is

β=102.826(1) °.

Embodiment 1.j

The fluoro-5-[2-(1-hydroxyl of 1-ethyl-3-[6--1-methyl-ethyl) pyrimidine-5-yl]-7-[(2R)-tetrahydrofuran (THF)-2-yl]-1 hydrogen-benzimidazolyl-2 radicals-yl] preparation of mesylate (24) of urea

By methylsulfonic acid (2.392mL, 36.89mmol, Sigma-Aldrich471356) join at methylene dichloride (60mL, J.T.Baker931533) and dehydrated alcohol (15mL, Pharmco-AAPER111000200) the fluoro-5-[2-(1-hydroxyl of the 1-ethyl-3-[6-in-1-methyl-ethyl) pyrimidine-5-yl]-7-[(2R)-tetrahydrofuran (THF)-2-yl]-1 hydrogen-benzimidazolyl-2 radicals-yl] stirred suspension of urea (23) (15.05g, 35.13mmol).In stirring at room until observe clear soln.With approximately slowly adding hexane (300mL) in 1 hour and collecting solid precipitation by filtering (using the qualitative #3 filter paper of Whatman on Whatman GF/F glass fiber filter paper).40 ℃ in vacuum oven drying under reduced pressure spend the night (with calcium sulfate and potassium hydroxide drying) produce the 24(13.46g of the solid that is white in color, 99+%HPLC purity, 99+%ee).Analytical chirality HPLC retention time is 8.6 minutes,

4.6x250mm post is used CH with 1mL/ minute flow velocity at 30 ℃ ₂Cl ₂/ MeOH/TEA(60/40/0.1) wash-out.The white solid 24(4.36g of second batch harvest, 98%HPLC purity, 99+%ee) from filtrate, obtain.LCMS(10-90%CH ₃CN/ water gradient elution C18 post 5 minutes, use formic acid properties-correcting agent) M+1:429.58 (2.03min). ¹H?NMR(300MHz,MeOD)δ9.00(d,J=1.6Hz,2H),7.67(d,J=6.1Hz,1H),5.39(t,J=7.7Hz,1H),4.30(dd,J=14.9,6.9Hz,1H),4.03(dd,J=14.8,7.7Hz,1H),3.40–3.31(m,2H),2.72(s,3H),2.70–2.60(m,1H),2.21–2.08(m,2H),1.98–1.84(m,1H),1.65(s,6H),1.22(t,J=7.2Hz,3H)ppm。

Embodiment 1.k

The fluoro-5-[2-(1-hydroxyl of 1-ethyl-3-[6--1-methyl-ethyl) pyrimidine-5-yl]-7-tetrahydrofuran (THF)-2-base-1 hydrogen-benzimidazolyl-2 radicals-yl] preparation of urea

To 2-[5-(4; the fluoro-3-tetrahydrofuran (THF) of 5-diamino-2--2-base-phenyl) pyrimidine-2-base] propan-2-ol (7.220g; 21.72mmol) and 1-ethyl-3-(N-(ethyl carbamyl)-C-methyl sulfo--carbon imino-) urea (6.054g; 26.06mmol; CB Research and Development) 1; 4-diox (36.1mL; Sigma-Aldrich360481) in the solution in, add pH3.5 damping fluid (72.2mL), described pH3.5 damping fluid is by being dissolved in the water-soluble H of 1N by NaOAc trihydrate (5.32g) ₂SO ₄(37mL) preparation in.Reaction mixture stirs spend the night (HPLC shows conversion fully) under refluxing, cool to room temperature, and be poured into water-soluble saturated NaHCO by part (foaming) ₃(144mL), provide pH8-9.The mixture that produces was stirred 20 minutes, and by solid collected by filtration, water fully washs to neutral pH, and subsequently with the washing of EtOH less amount ground.The solid drying under reduced pressure, produce beige solid (7.9g, 99%HPLC purity).LCMS(10-90%CH ₃CN/ water gradient elution C18 post 5 minutes, use formic acid properties-correcting agent) M+1:429.45 (2.03min).The HPLC retention time is 3.89 minutes (YMC ODS-AQ150x3.0mm post 10-90%CH ₃CN/ water gradient elution 8 minutes, with 0.1%TFA properties-correcting agent and 1mL/ minute flow velocity).

The preparation of form I

Embodiment 1.l

(R) the fluoro-5-[2-(1-hydroxyl of-1-ethyl-3-[6--1-methyl-ethyl) pyrimidine-5-yl]-7-(tetrahydrofuran (THF)-2-yl)-1 hydrogen-benzimidazolyl-2 radicals-yl] chiral chromatography of urea separates

The upper fluoro-5-[2-(1-hydroxyl of the 1-ethyl-3-[6--1-methyl-ethyl of resolving of post (by Chiral Technologies)) pyrimidine-5-yl]-7-tetrahydrofuran (THF)-2-base-1 hydrogen-benzimidazolyl-2 radicals-yl] the racemize sample of urea (133.60g), at 25 ℃, use DCM/MeOH/TEA(60/40/0.1) wash-out, generation is the required enantiomorph (66.8g of off-white color solid, 99.8%HPLC purity, 99+%ee).Analytical chirality HPLC retention time be 7.7 minutes ( 4.6x250mm post, 1mL/ minute flow velocity, 30 ℃).Described solid suspension is in 2:1EtOH/Et ₂O(5 times of volume), stirred 10 minutes, filter and collect, use 2:1EtOH/Et ₂O washes and drying under reduced pressure produces white solid (60.6g). ¹H?NMR(300MHz,MeOD)δ8.95(d,J=1.6Hz,2H),7.45(d,J=6.5Hz,1H),5.38(br.s,1H),4.27(dd,J=14.9,7.1Hz,1H),4.01(dd,J=15.1,7.0Hz,1H),3.37–3.29(m,2H),2.55(br.s,1H),2.19–2.07(m,2H),2.02–1.82(br.s,1H),1.63(s,6H),1.21(t,J=7.2Hz,3H)ppm。

The preparation of form II

Embodiment 1.m

1mL THF is joined in 100mg6-fluorobenzene imidazolyl carbamide compound.Add the HCl of stoichiometric quantity to be the aqueous solution of 12M.Add the MTBE of 4mL and allow suspension equilibrate overnight under stirring at room.Filter, and by white solid under vacuum dry several hours.

The preparation of form III

Embodiment 1.n

Weigh up 100mg6-fluorobenzene imidazolyl carbamide compound and be dissolved in 200ml methylene chloride/methanol 1:1(v:v) mixture.With (pump program 2) on the Buchi B-90 nano-spray moisture eliminator of condenser with 100% spraying rate by described solution spray drying.Use 101 ℃ of nitrogen with 10L/ minute of temperature in to flow, the peak pressure 10psi of nitrogen and maximum CO ₂Pressure 15psi, reclaim the 55mg white solid.

With on the Buchi B-90 nano-spray moisture eliminator of condenser, carrying out spraying drying.The solution of described 6-fluorobenzene imidazolyl carbamide compound prepares and sprays according to following parameters in comprising methylene chloride/methanol (1:1) solvent systems.

The preparation of form IV

Embodiment 1.o

(R) the fluoro-5-[2-(1-hydroxyl of-1-ethyl-3-[6--1-methyl-ethyl) pyrimidine-5-yl]-7-(tetrahydrofuran (THF)-2-yl)-1 hydrogen-benzimidazolyl-2 radicals-yl] preparation of mesylate of urea

At methylene dichloride (22.8mL, Sigma-Aldrich270997) and the fluoro-5-[2-(1-hydroxyl of (the R)-1-ethyl-3-[6-in dehydrated alcohol (2.5mL)-1-methyl-ethyl) pyrimidine-5-yl]-7-(tetrahydrofuran (THF)-2-yl)-1 hydrogen-benzimidazolyl-2 radicals-yl] urea (2.530g, 5.905mmol) stirred suspension with ice-water bath cooling.Add methylsulfonic acid (0.402mL, 6.20mmol, Sigma-Aldrich471356), remove cooling bath, and stirring at room 10 minutes.Mixture at 30 ℃ of oil that are condensed into stiff, then slowly joins the Et of stirring in rotatory evaporator ₂In O, and use CH ₂Cl ₂Resistates is flushed in ether.Stir the precipitation of viscosity until it is decomposed into the paste solid, by filtration, collect described solid, use Et ₂O washes, and drying under reduced pressure generation off-white color solid (2.85g, 99%HPLC purity, 99+%ee).LCMS(10-90%CH ₃CN/ water gradient elution C18 post 5 minutes, use formic acid properties-correcting agent) M+1:429.45 (2.49min).The HPLC retention time is 3.86 minutes (YMC ODS-AQ150x3.0mm post 10-90%CH ₃CN/ water gradient elution 8 minutes, with 0.1%TFA properties-correcting agent and 1mL/ minute flow velocity).

4.6x250mm on post, use DCM/MeOH/TEA(60/40/0.1) with 1mL/ minute flow velocity at 30 ℃ of wash-outs, analytical chirality HPLC retention time is 7.8 minutes. ¹HNMR(300MHz,MeOD)δ8.99(d,J=1.6Hz,2H),7.67(d,J=6.1Hz,1H),5.38(t,J=7.7Hz,1H),4.30(dd,J=15.0,6.9Hz,1H),4.02(dd,J=14.8,7.6Hz,1H),3.38–3.30(m,2H),2.73(s,3H),2.70–2.60(m,1H),2.20–2.07(m,2H),1.99–1.84(m,1H),1.64(s,6H),1.22(t,J=7.2Hz,3H)ppm。

Embodiment 1.p

Stability data

The sulfonate of finding 6-fluorobenzene imidazolyl carbamide compound under 25 ℃/60%RH during at week age point chemically and physically unsettled, and when t=2 is all, be chemically unsettled under being stored in 40 ℃/atmospheric environment.

Free alkali 6-fluorobenzene imidazolyl carbamide compound under whole conditions of storage (25 ℃/60%RH, 40 ℃/atmospheric environment, and 40 ℃/75%RH) one-month period during point chemically and physically stable.In the XRPD pattern, observe little variation, but think it is the form the same with the zero-time (t=0) generally.

The hydrochloride of 6-fluorobenzene imidazolyl carbamide compound under whole conditions of storage (25 ℃/60%RH, 40 ℃/atmospheric environment, and 40 ℃/75%RH) one-month period during point chemically and physically stable.

Embodiment 2

Enzymology

In the experiment that the enzyme inhibition activity of the application's compound can be described hereinafter, measure:

DNA gyrase atpase assay method

To, by the ADP production of pyruvate kinase/serum lactic dehydrogenase and the oxidation phase coupling of NADH, measure the ATP hydrolytic activity of streptococcus aureus DNA gyrase.This method was before described (Tamura and Gellert, 1990, J.Biol.Chem., 265,21342).

At 30 ℃, containing 100mM TRIS pH7.6,1.5mM MgCl ₂, 150mM KCl buffered soln in carry out the atpase assay method.Coupling system contains final concentration 2.5mM phosphoenolpyruvic acid, 200 μ M Reduced nicotinamide-adenine dinucleotides (NADH), 1mM DTT, 30ug/ml pyruvate kinase and 10ug/ml serum lactic dehydrogenase.The DMSO solution (3% final concentration) that adds described enzyme (90nM final concentration) and compound.Allow reaction mixture to hatch 10 minutes at 30 ℃.By the final concentration that adds ATP to 0.9mM, make to react initial, and in 340 nanometers, monitor the NADH rate of disappearances through the process of 10 minutes.By speed, with respect to the distribution plan of concentration, determine K _iAnd IC ₅₀Value.

The inhibition of table 3. streptococcus aureus DNA gyrase

Selected compounds K _i(nM)

Compound 23* 9

* can prepare by compound 23 as described in top embodiment 1.i.

DNA Topo IV atpase assay method

By the conversion coupling of the ATP to ADP of streptococcus aureus TopoIV enzyme conversion with NADH to NAD+, and by the progress of the reaction of the absorbancy measure of the change at the 340nm place.Make TopoIV(64nM) together with selected compounds (final 3%DMSO), in damping fluid, at 30 ℃, hatched 10 minutes.Damping fluid is comprised of 100mM Tris7.5,1.5mM MgCl2,200mMK glutaminate, 2.5mM phosphoenolpyruvic acid, 0.2mM NADH, 1mM DTT, 5 μ g/mL linearizing DNA, 50 μ g/mL BSA, 30 μ g/mL pyruvate kinases and 10 μ g/mL serum lactic dehydrogenases (LDH).With ATP, make to react initial, and on Molecular Devices SpectraMAX plate reader 30 ℃ of continuous monitoring speed 20 minutes.From speed, with respect to the graphic representation of the concentration of selected compounds, measure and suppress constant (Ki) and IC ₅₀, inhibitor is fitted to Morison formula for combining closely.

The inhibition of table 4. streptococcus aureus DNA TopoIV enzyme

Selected compounds K _i(nM)

Compound 23 12

Embodiment 3

Sensitivity tests in liquid medium within

By the sensitivity tests in liquid medium within, test the antimicrobial acivity of compound of the present invention.This type of assay method can be carried out in the guilding principle of the up-to-date CLSI file of managing this type of practice: " M07-A8Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; Approval standards-8th edition (2009) ".Other publications for example " Antibiotics in Laboratory Medicine " (being edited the Williams of publisher and Wilkins, 1996 by V.Lorian) provide the elementary practice technology in the test of laboratory microbiotic.The specified scheme of using is as follows:

Scheme #1: the mensuration of the gyrase MIC of the compound of use Microdilution meat soup method

Material:

Round bottom 96 hole microtiter plates (Costar3788)

Mueller Hinton II agar plate (MHII; The BBL pre-mixing)

Mueller Hinton II liquid meat soup (MHII; The BBL pre-mixing)

BBL?Prompt?Inoculation?System（Fisher?B26306）

Test reading mirror (Fisher)

The agar plate of streak culture bacterium to single bacterium colony, fresh preparation

Aseptic DMSO

Human serum (U.S.Biologicals S1010-51)

Cracking horse blood (Quad Five270-100)

Resazurin 0.01%

Sprague Dawley rat blood serum (U.S.Biologicals1011-90B or Valley BioMedical AS3061SD)

The mice serum (Valley BioMedical AS3054) that merges

Bacterial strain (substratum, meat soup and agar):

1. streptococcus aureus ATCC#29213

a.MHII

The b.MHII+50% human serum

The c.MHII+50% rat blood serum

The d.MHII+50% mice serum

2. streptococcus aureus ATCC#29213GyrB T173I(MHII)

3. streptococcus aureus, JMI collects bacterial strain; Referring to table 9(MHII)

4. staphylococcus epidermidis, JMI collects bacterial strain; Referring to table 9(MHII)

5. enterococcus faecalis ATCC#29212(MHII+3% cracking horse blood)

6. faecium ATCC#49624(MHII+3% cracking horse blood)

7. enterococcus faecalis, JMI collects bacterial strain; Referring to table 9(MHII+3% cracking horse blood)

8. faecium, JMI collects bacterial strain; Referring to table 9(MHII+3% cracking horse blood)

9. streptococcus pneumoniae ATCC#10015(MHII+3% cracking horse blood)

10. streptococcus pneumoniae, JMI collects bacterial strain; Referring to table 9(MHII+3% cracking horse blood)

11. Beta-hemolytic streptococcus, group A, B, C, G) JMI collects bacterial strain; Referring to table 9(MHII+3% cracking horse blood)

12. bacillus cereus (Bacillus cereus) ATCC 10987(MHII)

13. bacillus cereus ATCC 14579(MHII)

14. subtilis (Bacillus subtilis) ATCC 6638(MHII)

15. subtilis (168) ATCC 6051(MHII)

Inoculum preparation (for all bacterial strains except streptococcus aureus+50% serum):

1. use BBL Prompt test kit, in the culture of growing, select 5 large or bacterium colonies 10 little abundant separation on suitable nutrient agar as implied above, and the 1mL Sterile Saline that provides in test kit is provided.

2. by hole vortex～30 second, providing～10 ⁸The suspension of cell/mL.Actual density can be paved plate by the dilution by this suspension and be confirmed.

3. for every flat board of tested compound, by the 0.15mL cell is transferred to 15mL(～10 ⁶In the aseptic meat soup (or vide infra) of cell/mL), by suspension 1/100 dilution, vortex mixed subsequently.If detect 8 kinds of compounds of 1 block of compound with upper flat plate (>), comprise compound 23 or 24, volume can correspondingly increase so.

A. for enterococcus faecalis, faecium and streptococcus pneumoniae: use 14.1mL MHII+0.9mL cracking horse blood.

4. use 50 μ l cell (～5x10 ⁴Cell) inoculate each microtiter well (vide infra) that contains 50 μ l dilute in meat soup medicine.

Drug dilution, inoculation, MIC measure:

1. usually prepared with 12.8mg/mL concentration by all medicines/compound stoste in 100%DMSO.

2. medicine/compound stoste is diluted to the required final concentration of 200x in 50 μ L DMSO.If the initial concentration of MICs is 8 μ g/mL final concentrations, need so 6.25 μ L stostes+43.75 μ L DMSO.Start the dividing of the 1st row that each 200x stoste is placed in to new 96 hole microtiter plates.

3. 25 μ L DMSO are added in the 2-12 row of all row of the microtiter plate that contains 200x compound stoste, and from the 1st row until the 11st row serial dilution 25 μ L change the rifle head after every row.That is, 25 μ L compounds+25 μ L DMSO=2x extent of dilution.In serial end, stay " without compound " DMSO hole for contrast.

4. for every kind of tested bacterial strain (except streptococcus aureus+50% human serum), use the Matrix transfer pipet to prepare two microtiter plates with 50 μ L MHII meat soups.

5. before adding 50 μ l cells, 0.5 each extent of dilution of μ L (w/Matrix autospencer) is transferred in 50 μ L substratum/microtiter wells.After 1/200 was diluted in substratum+cell, the common initial concentration of compound was that the row of 8 μ g/mL-compound concentrations leap microtiter plates reduces with the 2x step-length.All MICs complete in duplicate.

The cell suspension (referring to above) of porose use 50 μ l dilutions be seeded to the final volume of 100 μ l.

7. after adding inoculum, with manual multichannel pipettor, fully mix each hole; Drug level uses identical rifle head from low to high on the same microtiter plate.

8. flat board was hatched 18 hours at 37 ℃ at least.

9. after 18 hours, with the test reading mirror, check flat board, and, when not observing growth (optically clear in hole), MIC is recorded as to the minimum concentration of medicine.

The preparation of streptococcus aureus+50% human serum, streptococcus aureus+50% rat blood serum or streptococcus aureus+50% mice serum.

1. by making up 15mL MHII+15mL Ren Xue Qing – 30mL altogether, preparation 50% blood serum medium.When test surpasses a kind of compound flat board, with the 30mL increment, increase volume.

2. use the identical BBLPrompt inoculum of streptococcus aureus ATCC#29213 as above, by the 0.15mL cell is transferred to 30mL(～5x10 ⁵Cell/mL) above interior 1/200 dilution of 50% human serum substratum of preparation, and vortex mixed.

3. be used in pack into all test holes of requisite number purpose microtiter plate of 100 μ L cells in 50% blood serum medium.

4. each diluted chemical compound degree of 0.5 μ L (w/Matrix autospencer) is transferred to 100 μ L cell/substratum.After 1/200 was diluted in substratum+cell, the common initial concentration of compound was that 8 μ g/mL-compound concentrations reduce with the capable 2x step-length of crossing over microtiter plate.All MICs complete in duplicate.

5. with manual multichannel pipettor, fully mix each hole; Drug level uses identical rifle head from low to high on the same microtiter plate.

6. flat board was hatched 18 hours at 37 ℃ at least.After hatching, 25 μ L0.01% resazurins are added to each hole, and continued to hatch other at least 1 hour or until resazurin variable color at 37 ℃.

7. with the test reading mirror, check flat board, and record MIC.When using resazurin, the color of dyestuff becomes bright pink colour from mazarine in the hole without growth.The minimum concentration that dyestuff is changed into to the medicine of pink colour is MIC.

Scheme #2: use the compound of Microdilution meat soup method to measure for gram-negative gyrase MIC

Material:

Round bottom 96 hole microtiter plates (Costar3788)

Mueller Hinton II agar plate (MHII; The BBL pre-mixing)

Mueller Hinton II liquid meat soup (MHII; The BBL pre-mixing)

BBL?Prompt?Inoculation?System（Fisher?b26306）

Test reading mirror (Fisher)

The agar plate of streak culture bacterium to single bacterium colony, fresh preparation

Aseptic DMSO

(the MHII substratum is for whole for bacterial strain; Meat soup and agar):

1. intestinal bacteria ATCC # 25922

2. intestinal bacteria, JMI collects bacterial strain, referring to table 9

3. intestinal bacteria AG100 WT

4. intestinal bacteria AG100 tolC

5. Acinetobacter baumannii (Acinetobacter baumannii) ATCC # BAA-1710

6. Acinetobacter baumannii ATCC # 19606

7. Acinetobacter baumannii, JMI collects bacterial strain, referring to table 9

8. Klebsiella pneumonia (Klebsiella pneumoniae) ATCC # BAA-1705

9. Klebsiella pneumonia ATCC # 700603

10. Klebsiella pneumonia, JMI collects bacterial strain, referring to table 9

11. moraxelle catarrhalis ATCC# 25238

12. moraxelle catarrhalis ATCC# 49143

13. moraxelle catarrhalis, JMI collects bacterial strain, referring to table 9

14. hemophilus influenzae ATCC 49247

15. hemophilus influenzae (Rd1 KW20) ATCC 51907

16. hemophilus influenzae Rd0894(AcrA-)

17. hemophilus influenzae, JMI collects bacterial strain, referring to table 9

18. Pseudomonas aeruginosa PAO1

19. Pseudomonas aeruginosa, JMI collects bacterial strain, referring to table 9

20. Proteus mirabilis (Proteus mirabilis), JMI collects bacterial strain, referring to table 9

21. enterobacter cloacae (Enterobacter cloacae), JMI collects bacterial strain, referring to table 9

22. stenotrophomonas maltophilia (Stenotrophomonas maltophilia) ATCC BAA-84

23. stenotrophomonas maltophilia ATCC13637

The inoculum preparation:

1. use BBL Prompt test kit, from the culture of growing at nutrient agar, selecting 5 large or bacterium colonies 10 little abundant separation, and be inoculated into the next 1mL Sterile Saline of test kit.

2. by hole vortex～30 second, providing～10 ⁸The suspension of cell/mL.Actual density can be paved plate by the dilution by this suspension and be confirmed.

3. for every flat board of tested compound, by the 0.15mL cell is transferred to 15mL(～10 ⁶In the aseptic meat soup (vide infra) of cell/mL), by suspension 1/100 dilution, vortex mixed.If test 8 kinds of compounds of 1 block of compound with upper flat plate (>), comprise compound 23 or 24, so corresponding increase volume.

4. use 50 μ l cell (～5x10 ⁴Cell) inoculate each microtiter well (vide infra) that contains 50 μ l dilute in meat soup medicine.

Drug dilution, inoculation, MIC measure:

1. usually prepared with 12.8mg/mL concentration by all medicines/compound stoste in 100%DMSO.

2. medicine/compound stoste is diluted to the required final concentration of 200x in 50 μ L DMSO.If the initial concentration of MICs is 8 μ g/mL final concentrations, need so 6.25 μ L stostes+43.75 μ L DMSO.Start the dividing of the 1st row that each 200x stoste is placed in to new 96 hole microtiter plates.

3. 25 μ L DMSO are added in the 2-12 row of all row of the microtiter plate that contains 200x compound stoste, and from the 1st row until the 11st row serial dilution 25 μ L change the rifle head after every row.That is, 25 μ L compounds+25 μ L DMSO=2x extent of dilution.In serial end, stay " without compound " DMSO hole for contrast.

4. for every kind of tested bacterial strain, use the Matrix transfer pipet to prepare two microtiter plates with 50 μ L MHII meat soups.

5. before adding 50 μ l cells, 0.5 each extent of dilution of μ L (w/Matrix autospencer) is transferred to 50 μ L substratum/microtiter wells.After 1/200 was diluted in substratum+cell, the common initial concentration of compound was that the row of 8 μ g/mL-compound concentrations leap microtiter plates reduces with the 2x step-length.All MICs complete in duplicate.

The cell suspension (referring to above) of porose use 50 μ l dilutions be seeded to the final volume of 100 μ l.

7. after adding inoculum, with manual multichannel pipettor, fully mix each hole; In identical microtiter plate Chinese traditional medicine concentration, use from low to high identical rifle head.

8. flat board was hatched 18 hours at 37 ℃ at least.

9. after 18 hours, with the test reading speculum, check flat board, and, when not observing growth (optically clear in hole), MIC is recorded as to the minimum concentration of medicine.

Scheme #3: use the gyrase MIC of the compound of agar dilution to measure

Material:

Culture dish 60x15mm(Thermo Scientific catalogue #12567100)

Centrifuge tube, 15mL(Costar)

BBL?Prompt?Inoculation?System（Fisher?B26306）

The agar plate of streak culture bacterium to single bacterium colony, fresh preparation

Aseptic DMSO

GasPak ^TMHatch container (BD catalogue #260672)

GasPak ^TMEZ Anaerobe containment system pouch (BD catalogue #260678)

GasPak ^TMEZ C02 containment system pouch (BD catalogue #260679)

GasPak ^TMEZ Campy containment system pouch (BD catalogue #260680)

Bacterial strain:

1. clostridium difficile ATCC BAA-1382;

2. clostridium difficile, CMI collects bacterial strain, referring to table 8

3. clostridium perfringens (Clostriudium perfringens), CMI collects bacterial strain, referring to table 8

4. bacteroides fragilis (Bacteroides fragilis) and Bacteroides bacterial classification (Bacteroides spp.), CMI collects bacterial strain, referring to table 8

5. Fusobacterium bacterial classification (Fusobacterium spp.), CMI collects bacterial strain, referring to table 8

6. Peptostreptococcus bacterial classification (Peptostreptococcus, spp.), CMI collects bacterial strain, referring to table 8

7. Prey is irrigated Pseudomonas bacterial classification (Prevotella spp.), and CMI collects bacterial strain, referring to table 8

8. neisseria gonorrhoeae ATCC 35541

9. neisseria gonorrhoeae ATCC 49226

10. neisseria gonorrhoeae, JMI collects bacterial strain, referring to table 8

11. Neisseria meningitidis, JMI collects bacterial strain, referring to table 8

Substratum preparation and growth conditions:

According to CLSI publication " M11-A7Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria; Approved Standard-Seventh Edition (2007) " growth medium recommended for each microbial species of preparation; except neisseria gonorrhoeae and Neisseria meningitidis, its substratum is according to " M07-A8Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; Approved Standard--Eighth Edition (2009) " is prepared.

Be down flat plate:

1. prepare the 100x medicine stoste of every kind of test compounds described in table 1.Use the 15mL centrifuge tube, every kind of medicine stoste of 100uL is added to 10mL fusing agar (being cooled in water-bath～55 ℃).Pipe is put upside down and mixed 2-3x, in the 60X15mm culture dish that impouring is subsequently marked respectively.

2. conventionally test concentration is: 0.002ug/mL – 16ug/mL(14 piece flat board).

3. topple over 4 without the medicine flat board: 2 as positive control, and 2 as aerobic contrast.

4. allow dull and stereotyped dry.Used the same day or stored and spend the night or reach 3 days 4 ℃ of storages at RT.

5. place correspondingly mark flat board for drug level and bacterial strain.

Need to remain the growth of the cell of anaerobic environment:

1. with all working that anerobe is carried out, all complete as far as possible rapidly; Being operated in before cell is got back to anaerobic chamber of carrying out in Biohazard Safety Equipment (being anaerobic environment) completes in less than 30 minutes.

2. use GasPak ^TMCulturing room realizes hatching of anerobe.Large box type culturing room (VWR90003-636) needs 2 anaerobism pouches (VWR90003-642), and high right cylinder formula culturing room (VWR90003-602) only needs 1 pouch.

Plating (carrying out in Biohazard Safety Equipment):

Every kind of bacterial strain is streak culture to agar plate as mentioned above 1..By required time and envrionment conditions (be anaerobism, trace aerobic etc.), hatch.

2. directly use the bacterium colony suspension method, with the fresh streak culture cell suspension by loopful, arrive～4mL0.9%NaCl ₂In and vortex.

3. suspension is adjusted to O.D. ₆₀₀0.05(5x10e7 cfu/mL).Vortex mixed.

4. the mixed culture of general～0.2mL adjustment is transferred to 96 hole flat boards.When≤5 kinds of bacterial strains of test, all bacterial strains are arranged together in single file.When test > during 5 kinds of bacterial strains, bacterial strain is transferred in flat board, in single file, have and be no more than 5 kinds of bacterial strains.This is essential to coordinating small plate.

5. use multichannel pipettor, will be from every kind of bacterial strain points of 0.002mL of 96 hole flat boards of preparation on every MIC test slab.This causes～the 1x10e5cfu/ point.When the test clostridium difficile, bacterial strain is vacillated in groups when growth, yet the distance between multichannel pipettor point is enough far away, makes the cell of vacillating not damage the assay method result.

A. at first inoculate 2 without the medicine flat board, and other 2 without the medicine flat board after the MIC test slab finally the inoculation.The former and the latter serve as growth and inoculation contrast.Without the one flat plate in medicine contrast inoculation, together with the MIC flat board, under required atmospheric condition, hatch for every group, and one is arranged under aerobic soon to test the pollution of aerophil.When with strictly anaerobic bacterium or microaerobion strain operation, its growth of aerobic cultivation is negative.For the more visible growths of neisseria gonorrhoeae.

6. allow inoculum dry (short as far as possible time), upside down is hatched in GasPak subsequently, wherein has the pouch of suitable number.

The neisserial species at 37 ℃ at 5%CO ₂In environment, hatched 24 hours.

MIC measures:

After correct incubation time, check tested flat board, and on test slab be grown in appearance with the positive control flat board on growth phase than under the concentration that remarkable minimizing occurs, reading the MIC terminal.

Table 5: the diluted chemical compound degree that uses agar dilution to measure for MIC

* 1,600ug/ml=64ul(10mg/ml stoste)+336ul DMSO; The 400ul cumulative volume is with initial

* dissolves compound and dilutes in 100%DMSO

Scheme #4. measures program for the MIC of mycobacterium species

Material

Round bottom 96 hole microtiter plates (Costar3788) or similar

The dull and stereotyped sealing of film (PerkinElmer, TopSeal-A#6005250 or similar)

Middlebrook7H10 meat soup with 0.2% glycerine

Middlebrook7H10 agar with 0.2% glycerine

Middlebrook?OADC?Enrichment

Inoculum preparation for mycobacterium tuberculosis:

1. the freezing mycobacterium tuberculosis original seed of used preparation is stored in-70 ℃.Mycobacterium tuberculosis is grown in 7H10 meat soup+10%OADC, subsequently with 100Klett or 5x10 ⁷The concentration of cfu/ml is freezing,

2. by taking out the freezing original seed of 1ml and it being added to 19ml7H10 meat soup+10%OADC(final concentration 2.5x10 ⁶Cfu/ml), preparation 1:20 extent of dilution.

3. by this extent of dilution, prepare second 1:20 extent of dilution, take out 1ml and it is added to the fresh meat soup of 19ml.This is the final inoculum that adds in 96 hole flat boards.

Inoculum preparation for mycobacterium kansasii, mycobacterium avium, mycobacterium abscessus and Nocardia bacteria species:

1. the freezing Primary spawn thing of used preparation or with 10Klett or 5x10 ⁷/ ml concentration is grown in 7H10 meat soup fresh culture thing.

2. by taking out 1ml, cultivate original seed and it is added to 19ml7H10 meat soup (final concentration 2.5x10 ⁶Cfu/ml), preparation 1:20 extent of dilution.

3. by this extent of dilution, preparation 1:20 extent of dilution, take out 1ml and it added to the fresh meat soup of 19ml (final suspension).

Dull and stereotyped preparation:

1. mark flat board.

2. use the multiple tracks electric pipettor, add the institute that measures for MIC porose 50 μ l 7H10 meat soup+10%OADC.

3. the stoste (for example 1mg/ml concentration) for preparing medicine to be tested.

4. freezing stoste thawed and use the dilution of 7H10 meat soup+10%OADC, with the peak concentration that obtains working solution 4x test (for example final concentration 32 μ g/ml, the maximum concentration of test is 8 μ g/ml).By stoste, prepared by extent of dilution.Initial for the concentration with 1 μ g/ml, prepared with 4 μ g/ml by medicine, so initial concentration is 1 μ g/ml.Take out 25 μ l 1mg/ml stostes, and add 6.2ml meat soup.All dilutions of medicine all complete in meat soup.

5. the 4x working solution of 50 μ l is added in first hole of nominated bank.For all compounds to be tested, continue.Use the multiple tracks electric pipettor, mix 4X and serial dilution compound until the 11st hole.Discard residue 50 μ l.Use the 12nd hole as positive control.

6. at 37 ℃, hatch flat board, mycobacterium tuberculosis～18 day; Mycobacterium avium and mycobacterium kansasii～7 day; Nocardia and mycobacterium abscessus～4 day; Use diaphragm seal.

7. estimate reading and record result.MIC is recorded as the minimum concentration (optically clear in hole) of the medicine of wherein not observing growth.

Scheme #5. is for the scheme of mycobacterium tuberculosis seroconversion MIC assay method

Material and reagent:

Costar#3904 black side, flat 96 hole microtiter plates

Middlebrook7H9 meat soup (BD271310) with 0.2% glycerine

Middlebrook?OADC?Enrichment

Foetal calf serum

Catalase (Sigma C1345)

Dextrose

NaCl ₂

BBL?Prompt?Inoculation?System（Fisher?b26306）

Has streak culture bacteria Agr to single bacterium colony dull and stereotyped (Middlebrook7H11 with 0.2% glycerine and OADC enrichment)

Aseptic DMSO

The substratum preparation:

1. for the MICs of seroconversion, need three kinds of different culture medias, it all has the basis of 7H9+0.2% glycerine.All substratum and fill-in all carry out sterilizing before MICs be important.

The preparation hereinafter all substratum and according to as next part described in inoculate.Use every kind of substratum to test all compounds for Mtb.

A.7H9+0.2% glycerine+10%OADC(" standard " MIC substratum)

B.7H9+0.2% glycerine+2g/L dextrose+0.85g/L NaCl+0.003g/L catalase (0%FBS).

C. with the 2x7H9+0.2% glycerine of isopyknic foetal calf serum (50%FBS) combination+2g/L dextrose+0.85g/L NaCl+0.003g/L catalase.

The inoculum preparation:

1. use BBL Prompt, select 5-10 bacterium colony that fully separates, and be seeded in 1ml Sterile Saline next in test kit.Usually, the slow growth due to this organism in cultivation, dull and stereotyped had for two to three ages in week when for this assay method.

2. abundant vortex, supersound process 30 seconds in water-bath subsequently, provide～10 ⁸The suspension of cell/ml.Actual density can be paved plate by the dilution by this suspension and be confirmed.

3. by 1/200 dilution BBL Prompt suspension (for example: the 0.2ml cell is transferred to the 40ml substratum), at three kinds of substratum preparations, prepare inoculum in separately, to obtain～10 ⁶The initiator cell density of cell/ml.

4. use 100 μ l cell (～5x10 ⁴Cell) inoculate each microtiter well (vide infra) that contains the medicine of 1 μ l in DMSO.

Drug dilution, inoculation, MIC measure:

1. prepared with 10mM by control drug stoste vazadrine and Vulkamycin. PA-93 in 100%DMSO, and in 50%DMSO and 100%DMSO, with 1mM, prepared respectively by Ciprofloxacin and Rifampin.The Xi Shi Du – of preparation is assigned to 100 μ L stostes in the first row of 96 hole flat boards.For every kind of compound, cross over row preparation 11 steps, 2 times of serial dilutions, it passes through 50 μ l in 50 μ l DMSO the 1st column jump to the 2 row.Continuation by 50 μ l from the 2nd column jump until the 11st row when every row, mix simultaneously and replacing rifle head.Stay only have DMSO the 12nd row in contrast.

2. before adding 100 μ l cells, 1 each extent of dilution of μ l is transferred in empty microtiter well.After in being diluted to substratum+cell, the initial concentration of vazadrine and Vulkamycin. PA-93 is 100 μ M; After in being diluted to substratum+cell, the initial concentration of Ciprofloxacin and Rifampin is 10 μ M.The row that compound concentration is crossed over microtiter plate with the 2x moved further reduces.Separately under condition, all MICs complete in duplicate at three kinds of substratum.

3. the test group of compound is usually with 10mM and 50 μ L volumes.

4. use multichannel pipettor, take out autonomous flat board every row all volumes and transfer in the first row of new 96 hole microtiter plates.Every row compound on main flat board is repeated to aforesaid operations, transfer in the 1st row of new 96 hole flat boards.

5. as described above for control compound, use DMSO as thinner, to generate 2 times, 11 extent of dilution of every kind of compound.In all cases, stay and only have the 12nd of DMSO to be listed as for contrast.In case all dilutions all complete, just as for control compound, completed, before adding 100 μ l cells, then 1 each extent of dilution of μ l is transferred in empty microtiter well.

The porose cell suspension inoculation (referring to above) of all using 100 μ l dilutions.

7. after inoculum adds, by the side of chucked flat board, mix dull and stereotyped.

8. flat board was hatched 9 days in 37 ℃ of culturing room of humidification.

9. in the time of 9 days, the aseptic resazurin of 25 μ l0.01% is added in each hole.Exciting 492nm, emission 595nm place to measure background fluorescence, and flat board is put back to incubator and treated other 24 hours.

10. after 24 hours, exciting 492nm, emission 595nm place to measure the fluorescence in each hole.

11. the inhibition percentage calculation of given compound is as follows: suppress per-cent=100-([hole fluorescence-average background fluorescence]/[DMSO contrast-average background fluorescence] x100).For all three kinds of culture medium condition, carry out the MICs scoring, it is under given culture medium condition, to suppress the minimum compound concentration that resazurin reduces (" %-inhibition ") signal >=70%.

Table 6 shows the result about the MIC assay method of the mesylate of benzimidazolyl-carbamide compound of the present invention.

In table 6 and rear continued and embodiment, " compound 24 " is that the mesylate of compound 23 also can be according to above embodiment 1.j preparation.This be with embodiment above in the identical numbering for the identification of described compound used.

The MIC value of table 6 – compound 24

Bacterial strain/specified conditions	Scheme	Compound 24
			Streptococcus aureus ATCC29213	1	0.021
Streptococcus aureus ATCC29213 end user serum	1	0.15
			Streptococcus aureus ATCC29213 uses rat blood serum	1	0.18
Streptococcus aureus ATCC29213 uses mice serum	1	0.5
			Streptococcus aureus ATCC29213GyrB T173I	1	0.3
Enterococcus faecalis ATCC29212, use cracking horse blood	1	0.028
			Faecium ATCC49624 uses cracking horse blood	1	0.11
Faecium ATCC49624	1	0.11
			Streptococcus pneumoniae ATCC10015, use cracking horse blood	1	0.01
Bacillus cereus ATCC 10987	1	0.031
			Bacillus cereus ATCC14579	1	0.031
Subtilis ATCC6638	1	2
			Subtilis (168) ATCC6051	1	4
Clostridium difficile ATCC BAA-1382	3	0.38
			Hemophilus influenzae ATCC49247	2	0.5
Hemophilus influenzae (Rd1 KW20) ATCC51907	2	1.3
			Hemophilus influenzae Rd0894(AcrA-)	2	0.041
Moraxelle catarrhalis ATCC25238	2	≤0.016
			Moraxelle catarrhalis ATCC49143	2	≤0.016
Neisseria gonorrhoeae ATCC35541	3	0.42
			Neisseria gonorrhoeae ATCC49226	3	1
Intestinal bacteria AG100WT	2	4
			Intestinal bacteria AG100tolC	2	0.063
Intestinal bacteria ATCC25922	2	12
			Intestinal bacteria CHE30	2	8
Intestinal bacteria CHE30tolC	2	0.125
			Intestinal bacteria MC4100	2	>16
Intestinal bacteria MC4100tolC	2	0.25

Bacterial strain/specified conditions	Scheme	Compound 24
			Klebsiella pneumonia ATCC700603	2	16
Klebsiella pneumonia ATCC BAA-1705	2	12
			Acinetobacter baumannii ATCC19606	2	8
Acinetobacter baumannii ATCC BAA-1710	2	6
			Pseudomonas aeruginosa PAO1	2	>16
Pseudomonas aeruginosa PAO750	2	0.25
			Stenotrophomonas maltophilia ATCC BAA-84	2	>8
Stenotrophomonas maltophilia ATCC13637	2	>8
			Mycobacterium avium 103	4	0.18
Mycobacterium avium Far	4	0.23
			Mycobacterium avium 3404.4	4	0.23
Nocardia caviae (Nocardia caviae) 2497	4	0.125
			Nocardia asteroides (N.asteroids) 2039	4	1
New Nocardia (N.nova) 10	4	1
			Mycobacterium kansasii 303	4	0.03
Mycobacterium kansasii 316	4	0.06
			Mycobacterium kansasii 379	4	<0.015
Mycobacterium tuberculosis H37Rv ATCC25618	4	0.015
			Mycobacterium tuberculosis Erdman ATCC35801	4	0.06
Mycobacterium tuberculosis Erdman ATCC35801	5	0.03
			Mycobacterium tuberculosis Erdman ATCC35801 mice serum	5	0.5
Mycobacterium abscessus BB2	4	1
			Mycobacterium abscessus MC6005	4	1
Mycobacterium abscessus MC5931	4	0.5
			Mycobacterium abscessus MC5605	4	1.5
Mycobacterium abscessus MC6025	4	0.75
			Mycobacterium abscessus MC5908	4	1.5
Mycobacterium abscessus BB3	4	0.5
			Mycobacterium abscessus BB4	4	2
Mycobacterium abscessus BB5	4	0.5
			Mycobacterium abscessus MC5922	4	0.25
Mycobacterium abscessus MC5960	4	0.5
			Mycobacterium abscessus BB1	4	2
Mycobacterium abscessus MC5812	4	1
			Mycobacterium abscessus MC5901	4	1
Mycobacterium abscessus BB6	4	0.5
			Mycobacterium abscessus BB8	4	0.5

Bacterial strain/specified conditions	Scheme	Compound 24
			Mycobacterium abscessus MC5908	4	1
Mycobacterium abscessus LT949	4	1
			Mycobacterium abscessus BB10	4	0.015
Mycobacterium abscessus MC6142	4	0.5
			Mycobacterium abscessus MC6136	4	0.5
Mycobacterium abscessus MC6111	4	0.5
			Mycobacterium abscessus MC6153	4	1

Table 7 shows the MIC90 assay method result about selected compounds of the present invention.

The MIC90 value of the experimental subjects group of table 7 – selected compounds and Gram-positive, Gram-negative and anaerobic pathogens

In following table 8, term " CMI " representative is positioned at Wilsonville, the Clinical Microbiology Institute of Oregon.

Table 8: be used to the experimental subjects group of the anaerobe body that generates the MIC90 data

In table 9, term " JMI " representative is positioned at North Liberty, the Jones Microbiology Institute of Iowa.

Table 9: be used to the Gram-positive that generates the MIC90 data and the experimental subjects group of gram negative organism

JMI isolate #	JMI organism coding	Organism
			394	ACB	Acinetobacter baumannii
2166	ACB	Acinetobacter baumannii
			3060	ACB	Acinetobacter baumannii
3170	ACB	Acinetobacter baumannii
			9328	ACB	Acinetobacter baumannii
9922	ACB	Acinetobacter baumannii
			13618	ACB	Acinetobacter baumannii
14308	ACB	Acinetobacter baumannii

JMI isolate #	JMI organism coding	Organism
			17086	ACB	Acinetobacter baumannii
17176	ACB	Acinetobacter baumannii
			30554	ACB	Acinetobacter baumannii
32007	ACB	Acinetobacter baumannii
			1192	ECL	Enterobacter cloacae
3096	ECL	Enterobacter cloacae
			5534	ECL	Enterobacter cloacae
6487	ECL	Enterobacter cloacae
			9592	ECL	Enterobacter cloacae
11680	ECL	Enterobacter cloacae
			12573	ECL	Enterobacter cloacae
12735	ECL	Enterobacter cloacae
			13057	ECL	Enterobacter cloacae
18048	ECL	Enterobacter cloacae
			25173	ECL	Enterobacter cloacae
29443	ECL	Enterobacter cloacae
			44	EF	Enterococcus faecalis
355	EF	Enterococcus faecalis
			886	EF	Enterococcus faecalis
955	EF	Enterococcus faecalis
			1000	EF	Enterococcus faecalis
1053	EF	Enterococcus faecalis
			1142	EF	Enterococcus faecalis
1325	EF	Enterococcus faecalis
			1446	EF	Enterococcus faecalis
2014	EF	Enterococcus faecalis
			2103	EF	Enterococcus faecalis
2255	EF	Enterococcus faecalis
			2978	EF	Enterococcus faecalis
2986	EF	Enterococcus faecalis
			5027	EF	Enterococcus faecalis
5270	EF	Enterococcus faecalis
			5874	EF	Enterococcus faecalis
7430	EF	Enterococcus faecalis
			7904	EF	Enterococcus faecalis
8092	EF	Enterococcus faecalis
			8691	EF	Enterococcus faecalis

JMI isolate #	JMI organism coding	Organism
			9090	EF	Enterococcus faecalis
10795	EF	Enterococcus faecalis
			14104	EF	Enterococcus faecalis
16481	EF	Enterococcus faecalis
			18217	EF	Enterococcus faecalis
22442	EF	Enterococcus faecalis
			25726	EF	Enterococcus faecalis
26143	EF	Enterococcus faecalis
			28131	EF	Enterococcus faecalis
29765	EF	Enterococcus faecalis
			30279	EF	Enterococcus faecalis
31234	EF	Enterococcus faecalis
			31673	EF	Enterococcus faecalis
115	EFM	Faecium
			227	EFM	Faecium
414	EFM	Faecium
			712	EFM	Faecium
870	EFM	Faecium
			911	EFM	Faecium
2356	EFM	Faecium
			2364	EFM	Faecium
2762	EFM	Faecium
			3062	EFM	Faecium
4464	EFM	Faecium
			4473	EFM	Faecium
4653	EFM	Faecium
			4679	EFM	Faecium
6803	EFM	Faecium
			6836	EFM	Faecium
8280	EFM	Faecium
			8702	EFM	Faecium
9855	EFM	Faecium
			10766	EFM	Faecium
12799	EFM	Faecium
			13556	EFM	Faecium
13783	EFM	Faecium
			14687	EFM	Faecium

JMI isolate #	JMI organism coding	Organism
			15268	EFM	Faecium
15525	EFM	Faecium
			15538	EFM	Faecium
18102	EFM	Faecium
			18306	EFM	Faecium
19967	EFM	Faecium
			22428	EFM	Faecium
23482	EFM	Faecium
			29658	EFM	Faecium
597	EC	Intestinal bacteria
			847	EC	Intestinal bacteria
1451	EC	Intestinal bacteria
			8682	EC	Intestinal bacteria
11199	EC	Intestinal bacteria
			12583	EC	Intestinal bacteria
12792	EC	Intestinal bacteria
			13265	EC	Intestinal bacteria
14594	EC	Intestinal bacteria
			22148	EC	Intestinal bacteria
29743	EC	Intestinal bacteria
			30426	EC	Intestinal bacteria
470	BSA	The A group streptococcus
			2965	BSA	The A group streptococcus
3112	BSA	The A group streptococcus
			3637	BSA	The A group streptococcus
4393	BSA	The A group streptococcus
			4546	BSA	The A group streptococcus
4615	BSA	The A group streptococcus
			5848	BSA	The A group streptococcus
6194	BSA	The A group streptococcus
			8816	BSA	The A group streptococcus
11814	BSA	The A group streptococcus
			16977	BSA	The A group streptococcus
18083	BSA	The A group streptococcus
			18821	BSA	The A group streptococcus
25178	BSA	The A group streptococcus
			30704	BSA	The A group streptococcus

JMI isolate #	JMI organism coding	Organism
			12	BSB	The B group streptococcus
10366	BSB	The B group streptococcus
			10611	BSB	The B group streptococcus
16786	BSB	The B group streptococcus
			18833	BSB	The B group streptococcus
30225	BSB	The B group streptococcus
			10422	BSC	The C group streptococcus
14209	BSC	The C group streptococcus
			29732	BSC	The C group streptococcus
8544	BSG	The G group streptococcus
			18086	BSG	The G group streptococcus
29815	BSG	The G group streptococcus
			147	HI	Hemophilus influenzae
180	HI	Hemophilus influenzae
			934	HI	Hemophilus influenzae
970	HI	Hemophilus influenzae
			1298	HI	Hemophilus influenzae
1819	HI	Hemophilus influenzae
			1915	HI	Hemophilus influenzae
2000	HI	Hemophilus influenzae
			2562	HI	Hemophilus influenzae
2821	HI	Hemophilus influenzae
			3133	HI	Hemophilus influenzae
3140	HI	Hemophilus influenzae
			3497	HI	Hemophilus influenzae
3508	HI	Hemophilus influenzae
			3535	HI	Hemophilus influenzae
4082	HI	Hemophilus influenzae
			4108	HI	Hemophilus influenzae
4422	HI	Hemophilus influenzae
			4868	HI	Hemophilus influenzae
4872	HI	Hemophilus influenzae
			5858	HI	Hemophilus influenzae
6258	HI	Hemophilus influenzae
			6875	HI	Hemophilus influenzae
7063	HI	Hemophilus influenzae
			7600	HI	Hemophilus influenzae

JMI isolate #	JMI organism coding	Organism
			8465	HI	Hemophilus influenzae
10280	HI	Hemophilus influenzae
			10732	HI	Hemophilus influenzae
10850	HI	Hemophilus influenzae
			11366	HI	Hemophilus influenzae
11716	HI	Hemophilus influenzae
			11724	HI	Hemophilus influenzae
11908	HI	Hemophilus influenzae
			12093	HI	Hemophilus influenzae
12107	HI	Hemophilus influenzae
			13424	HI	Hemophilus influenzae
13439	HI	Hemophilus influenzae
			13672	HI	Hemophilus influenzae
13687	HI	Hemophilus influenzae
			13792	HI	Hemophilus influenzae
13793	HI	Hemophilus influenzae
			14440	HI	Hemophilus influenzae
15351	HI	Hemophilus influenzae
			15356	HI	Hemophilus influenzae
15678	HI	Hemophilus influenzae
			15800	HI	Hemophilus influenzae
17841	HI	Hemophilus influenzae
			18614	HI	Hemophilus influenzae
25195	HI	Hemophilus influenzae
			27021	HI	Hemophilus influenzae
28326	HI	Hemophilus influenzae
			28332	HI	Hemophilus influenzae
29918	HI	Hemophilus influenzae
			29923	HI	Hemophilus influenzae
31911	HI	Hemophilus influenzae
			428	KPN	Klebsiella pneumonia
791	KPN	Klebsiella pneumonia
			836	KPN	Klebsiella pneumonia
1422	KPN	Klebsiella pneumonia
			1674	KPN	Klebsiella pneumonia
1883	KPN	Klebsiella pneumonia
			6486	KPN	Klebsiella pneumonia

JMI isolate #	JMI organism coding	Organism
			8789	KPN	Klebsiella pneumonia
10705	KPN	Klebsiella pneumonia
			11123	KPN	Klebsiella pneumonia
28148	KPN	Klebsiella pneumonia
			29432	KPN	Klebsiella pneumonia
937	MCAT	Moraxelle catarrhalis
			1290	MCAT	Moraxelle catarrhalis
1830	MCAT	Moraxelle catarrhalis
			1903	MCAT	Moraxelle catarrhalis
4346	MCAT	Moraxelle catarrhalis
			4880	MCAT	Moraxelle catarrhalis
6241	MCAT	Moraxelle catarrhalis
			6551	MCAT	Moraxelle catarrhalis
7074	MCAT	Moraxelle catarrhalis
			7259	MCAT	Moraxelle catarrhalis
7544	MCAT	Moraxelle catarrhalis
			8142	MCAT	Moraxelle catarrhalis
8451	MCAT	Moraxelle catarrhalis
			9246	MCAT	Moraxelle catarrhalis
9996	MCAT	Moraxelle catarrhalis
			12158	MCAT	Moraxelle catarrhalis
13443	MCAT	Moraxelle catarrhalis
			13692	MCAT	Moraxelle catarrhalis
13817	MCAT	Moraxelle catarrhalis
			14431	MCAT	Moraxelle catarrhalis
14762	MCAT	Moraxelle catarrhalis
			14842	MCAT	Moraxelle catarrhalis
15361	MCAT	Moraxelle catarrhalis
			15741	MCAT	Moraxelle catarrhalis
17843	MCAT	Moraxelle catarrhalis
			18639	MCAT	Moraxelle catarrhalis
241	GC	Neisseria gonorrhoeae
			291	GC	Neisseria gonorrhoeae
293	GC	Neisseria gonorrhoeae
			344	GC	Neisseria gonorrhoeae
451	GC	Neisseria gonorrhoeae
			474	GC	Neisseria gonorrhoeae

JMI isolate #	JMI organism coding	Organism
			491	GC	Neisseria gonorrhoeae
493	GC	Neisseria gonorrhoeae
			503	GC	Neisseria gonorrhoeae
521	GC	Neisseria gonorrhoeae
			552	GC	Neisseria gonorrhoeae
573	GC	Neisseria gonorrhoeae
			592	GC	Neisseria gonorrhoeae
25	NM	Neisseria meningitidis
			813	NM	Neisseria meningitidis
1725	NM	Neisseria meningitidis
			2747	NM	Neisseria meningitidis
3201	NM	Neisseria meningitidis
			3335	NM	Neisseria meningitidis
7053	NM	Neisseria meningitidis
			9407	NM	Neisseria meningitidis
10447	NM	Neisseria meningitidis
			12685	NM	Neisseria meningitidis
12841	NM	Neisseria meningitidis
			14038	NM	Neisseria meningitidis
1127	PM	Proteus mirabilis
			3049	PM	Proteus mirabilis
4471	PM	Proteus mirabilis
			8793	PM	Proteus mirabilis
10702	PM	Proteus mirabilis
			11218	PM	Proteus mirabilis
14662	PM	Proteus mirabilis
			17072	PM	Proteus mirabilis
19059	PM	Proteus mirabilis
			23367	PM	Proteus mirabilis
29819	PM	Proteus mirabilis
			31419	PM	Proteus mirabilis
1881	PSA	Pseudomonas aeruginosa
			5061	PSA	Pseudomonas aeruginosa
7909	PSA	Pseudomonas aeruginosa
			8713	PSA	Pseudomonas aeruginosa
14318	PSA	Pseudomonas aeruginosa
			14772	PSA	Pseudomonas aeruginosa

JMI isolate #	JMI organism coding	Organism
			15512	PSA	Pseudomonas aeruginosa
17093	PSA	Pseudomonas aeruginosa
			17802	PSA	Pseudomonas aeruginosa
19661	PSA	Pseudomonas aeruginosa
			29967	PSA	Pseudomonas aeruginosa
31539	PSA	Pseudomonas aeruginosa
			82	SA	Streptococcus aureus
99	SA	Streptococcus aureus
			138	SA	Streptococcus aureus
139	SA	Streptococcus aureus
			140	SA	Streptococcus aureus
141	SA	Streptococcus aureus
			142	SA	Streptococcus aureus
272	SA	Streptococcus aureus
			287	SA	Streptococcus aureus
354	SA	Streptococcus aureus
			382	SA	Streptococcus aureus
1112	SA	Streptococcus aureus
			1687	SA	Streptococcus aureus
1848	SA	Streptococcus aureus
			2031	SA	Streptococcus aureus
2159	SA	Streptococcus aureus
			2645	SA	Streptococcus aureus
3256	SA	Streptococcus aureus
			3276	SA	Streptococcus aureus
4044	SA	Streptococcus aureus
			4214	SA	Streptococcus aureus
4217	SA	Streptococcus aureus
			4220	SA	Streptococcus aureus
4231	SA	Streptococcus aureus
			4240	SA	Streptococcus aureus
4262	SA	Streptococcus aureus
			4370	SA	Streptococcus aureus
4665	SA	Streptococcus aureus
			4666	SA	Streptococcus aureus
4667	SA	Streptococcus aureus
			5026	SA	Streptococcus aureus

JMI isolate #	JMI organism coding	Organism
			5666	SA	Streptococcus aureus
6792	SA	Streptococcus aureus
			7023	SA	Streptococcus aureus
7461	SA	Streptococcus aureus
			7899	SA	Streptococcus aureus
7901	SA	Streptococcus aureus
			8714	SA	Streptococcus aureus
9374	SA	Streptococcus aureus
			9437	SA	Streptococcus aureus
10056	SA	Streptococcus aureus
			10110	SA	Streptococcus aureus
11379	SA	Streptococcus aureus
			11629	SA	Streptococcus aureus
11659	SA	Streptococcus aureus
			12788	SA	Streptococcus aureus
12789	SA	Streptococcus aureus
			13043	SA	Streptococcus aureus
13086	SA	Streptococcus aureus
			13721	SA	Streptococcus aureus
13742	SA	Streptococcus aureus
			13932	SA	Streptococcus aureus
14210	SA	Streptococcus aureus
			14384	SA	Streptococcus aureus
15428	SA	Streptococcus aureus
			15430	SA	Streptococcus aureus
17721	SA	Streptococcus aureus
			18688	SA	Streptococcus aureus
19095	SA	Streptococcus aureus
			20195	SA	Streptococcus aureus
22141	SA	Streptococcus aureus
			22689	SA	Streptococcus aureus
27398	SA	Streptococcus aureus
			29048	SA	Streptococcus aureus
29051	SA	Streptococcus aureus
			30491	SA	Streptococcus aureus
30538	SA	Streptococcus aureus
			25	SEPI	Staphylococcus epidermidis

JMI isolate #	JMI organism coding	Organism
			53	SEPI	Staphylococcus epidermidis
385	SEPI	Staphylococcus epidermidis
			398	SEPI	Staphylococcus epidermidis
701	SEPI	Staphylococcus epidermidis
			713	SEPI	Staphylococcus epidermidis
1381	SEPI	Staphylococcus epidermidis
			2174	SEPI	Staphylococcus epidermidis
2286	SEPI	Staphylococcus epidermidis
			2969	SEPI	Staphylococcus epidermidis
3417	SEPI	Staphylococcus epidermidis
			3447	SEPI	Staphylococcus epidermidis
4753	SEPI	Staphylococcus epidermidis
			7241	SEPI	Staphylococcus epidermidis
9366	SEPI	Staphylococcus epidermidis
			10665	SEPI	Staphylococcus epidermidis
11792	SEPI	Staphylococcus epidermidis
			12311	SEPI	Staphylococcus epidermidis
13036	SEPI	Staphylococcus epidermidis
			13227	SEPI	Staphylococcus epidermidis
13243	SEPI	Staphylococcus epidermidis
			13621	SEPI	Staphylococcus epidermidis
13638	SEPI	Staphylococcus epidermidis
			13800	SEPI	Staphylococcus epidermidis
14078	SEPI	Staphylococcus epidermidis
			14392	SEPI	Staphylococcus epidermidis
15007	SEPI	Staphylococcus epidermidis
			16733	SEPI	Staphylococcus epidermidis
18871	SEPI	Staphylococcus epidermidis
			23285	SEPI	Staphylococcus epidermidis
27805	SEPI	Staphylococcus epidermidis
			29679	SEPI	Staphylococcus epidermidis
29985	SEPI	Staphylococcus epidermidis
			30259	SEPI	Staphylococcus epidermidis
31444	SEPI	Staphylococcus epidermidis
			268	SPN	Streptococcus pneumoniae
1264	SPN	Streptococcus pneumoniae
			2482	SPN	Streptococcus pneumoniae

JMI isolate #	JMI organism coding	Organism
			2653	SPN	Streptococcus pneumoniae
2994	SPN	Streptococcus pneumoniae
			3123	SPN	Streptococcus pneumoniae
3124	SPN	Streptococcus pneumoniae
			4336	SPN	Streptococcus pneumoniae
4858	SPN	Streptococcus pneumoniae
			5606	SPN	Streptococcus pneumoniae
5881	SPN	Streptococcus pneumoniae
			5897	SPN	Streptococcus pneumoniae
5900	SPN	Streptococcus pneumoniae
			6051	SPN	Streptococcus pneumoniae
6216	SPN	Streptococcus pneumoniae
			6556	SPN	Streptococcus pneumoniae
7270	SPN	Streptococcus pneumoniae
			7584	SPN	Streptococcus pneumoniae
8479	SPN	Streptococcus pneumoniae
			8501	SPN	Streptococcus pneumoniae
9256	SPN	Streptococcus pneumoniae
			9257	SPN	Streptococcus pneumoniae
10246	SPN	Streptococcus pneumoniae
			10467	SPN	Streptococcus pneumoniae
10886	SPN	Streptococcus pneumoniae
			11217	SPN	Streptococcus pneumoniae
11228	SPN	Streptococcus pneumoniae
			11238	SPN	Streptococcus pneumoniae
11757	SPN	Streptococcus pneumoniae
			11768	SPN	Streptococcus pneumoniae
12121	SPN	Streptococcus pneumoniae
			12124	SPN	Streptococcus pneumoniae
12149	SPN	Streptococcus pneumoniae
			12767	SPN	Streptococcus pneumoniae
12988	SPN	Streptococcus pneumoniae
			13321	SPN	Streptococcus pneumoniae
13393	SPN	Streptococcus pneumoniae
			13521	SPN	Streptococcus pneumoniae
13544	SPN	Streptococcus pneumoniae
			13700	SPN	Streptococcus pneumoniae

JMI isolate #	JMI organism coding	Organism
			13704	SPN	Streptococcus pneumoniae
13822	SPN	Streptococcus pneumoniae
			13838	SPN	Streptococcus pneumoniae
14131	SPN	Streptococcus pneumoniae
			14413	SPN	Streptococcus pneumoniae
14744	SPN	Streptococcus pneumoniae
			14808	SPN	Streptococcus pneumoniae
14827	SPN	Streptococcus pneumoniae
			14835	SPN	Streptococcus pneumoniae
14836	SPN	Streptococcus pneumoniae
			15832	SPN	Streptococcus pneumoniae
17336	SPN	Streptococcus pneumoniae
			17343	SPN	Streptococcus pneumoniae
17349	SPN	Streptococcus pneumoniae
			17735	SPN	Streptococcus pneumoniae
18060	SPN	Streptococcus pneumoniae
			18567	SPN	Streptococcus pneumoniae
18595	SPN	Streptococcus pneumoniae
			19082	SPN	Streptococcus pneumoniae
19826	SPN	Streptococcus pneumoniae
			22174	SPN	Streptococcus pneumoniae
22175	SPN	Streptococcus pneumoniae
			27003	SPN	Streptococcus pneumoniae
28310	SPN	Streptococcus pneumoniae
			28312	SPN	Streptococcus pneumoniae
29890	SPN	Streptococcus pneumoniae
			29910	SPN	Streptococcus pneumoniae

Claims (24)

1. the solid form I of the compound of formula (I):

2. the solid form I of claim 1, is characterized in that X-ray powder diffraction pattern (XPRD) is when using Cu K _αDuring radiation measurement, comprise at least 3 approximate peak positions (2 θ angles ± 0.2), when XPRD from about 5 degree when approximately 2 θ of 38 degree collect, it is selected from 9.3,11.7,12.1,12.4,14.5,15.9,16.3,16.6,18.5,19.4,21.5,22.3,22.8,23.8,24.5,25.7,28.1,28.4,30.3 and 33.4.

3. the solid form I of claim 1, is characterized in that X-ray powder diffraction pattern (XPRD) is when using Cu K _αDuring radiation measurement, comprise at least 3 approximate peak positions (2 θ angles ± 0.2), when XPRD from about 5 degree when approximately 2 θ of 38 degree collect, it is selected from 9.3,16.6,18.5,19.4,21.5 and 25.7.

4. the solid form I of claim 3, is characterized in that as used Cu K _αRadiometric, be substantially similar to the X-ray powder diffraction pattern of Fig. 1.

5. the solid form I of claim 4, it is further characterized in that as measured with the differential scanning calorimetries of about 10 ℃ of/minute scanning temperature by temperature wherein, has approximately the endotherm(ic)peak of the starting temperature of 318 ℃.

6. method for the preparation of the crystallized form I of the compound of according to claim 1 formula (I), its be included in solvent systems suspend described free alkali solid material and separate described solid, described solvent systems comprises alcohol or ether.

7. the hydrochloride of the compound of formula (I):

8. the hydrochloride of claim 7, wherein said salt is form II solid form.

9. the hydrochloride of claim 8, wherein said form II solid are characterised in that X-ray powder diffraction pattern (XPRD) is when using Cu K _αDuring radiation measurement, comprise at least 3 approximate peak positions (2 θ angles ± 0.2), when XPRD from about 5 degree when approximately 2 θ of 38 degree collect, it is selected from 6.7,9.2,16.7,18.6,19.5,20.5,25.6 and 27.5.

10. the hydrochloride of claim 8, wherein said form II solid form are characterised in that as used Cu K _αRadiometric, be substantially similar to the X-ray powder diffraction pattern of Fig. 4.

11. it is as measured as the differential scanning calorimetries by with about 10 ℃ of/minute scanning temperature that the hydrochloride of claim 9, wherein said form II solid form are further characterized in that, solid form has starting temperature in the about endotherm(ic)peak of 252 ℃.

12. the method for the preparation of the solid form II of the hydrochloride of the compound of according to claim 8 formula (I), it comprises the free alkali of 6-fluorobenzene imidazolyl carbamide compound is suspended in the acid solvent mixture that comprises one or more ether solvents and water.

13. the 6-fluorobenzene of formula I the unbodied form III of imidazolyl carbamide compound:

14. the unbodied form III of the fluorobenzene of claim 13 imidazolyl carbamide compound, it is characterized in that having can not the identification diffraction peak broad halo be the use Cu K of feature _αThe X-ray powder diffraction pattern (XPRD) of radiation.

15. the method for the preparation of the unbodied form III of according to claim 13 6-fluorobenzene imidazolyl carbamide compound, it comprises the solution of the dry 6-fluorobenzene of freeze-drying, spraying drying, drum drying or pulses switch imidazolyl carbamide compound.

16. the amorphous form IV of the 6-fluorobenzene of formula I the mesylate of imidazolyl carbamide compound:

17. the amorphous form IV of the 6-fluorobenzene of claim 16 mesylate of imidazolyl carbamide compound, it is characterized in that having can not the identification diffraction peak broad halo be the use Cu K of feature _αThe X-ray powder diffraction pattern (XPRD) of radiation.

18. the hydrochloride of claim 8, wherein form II solid form is at 40 ℃ with relative humidity is high stablizes to 75% the time at least one month.

19. the solid form I of claim 2, wherein said solid is at 40 ℃ and relative humidity is high stablizes to 75% the time at least one month.

20. comprise according to claim 1 described compound and pharmaceutically acceptable carrier, adjuvant or vectorial pharmaceutical composition.

21. the method for progress, seriousness or effect that in control, treatment or minimizing patient's hospital or non-hospital, bacterium infects, it comprises to described patient uses pharmaceutical composition according to claim 20.

22. method according to claim 21, wherein said bacterium infect one or more the existence that is characterised in that in following: streptococcus pneumoniae (Streptococcus pneumoniae), staphylococcus epidermidis (Staphylococcus epidermidis), enterococcus faecalis (Enterococcus faecalis), streptococcus aureus (Staphylococcus aureus), clostridium difficile (Clostridium difficile), moraxelle catarrhalis (Moraxella catarrhalis), neisseria gonorrhoeae (Neisseria gonorrhoeae), Neisseria meningitidis (Neisseria meningitidis), the multiple disease (Mycobacterium avium complex) of mycobacterium avium, mycobacterium abscessus (Mycobacterium abscessus), mycobacterium kansasii (Mycobacterium kansasii), mycobacterium buruli (Mycobacterium ulcerans), Chlamydophila pneumoniae (Chlamydophila pneumoniae), chlamydia trachomatis (Chlamydia trachomatis), hemophilus influenzae (Haemophilus influenzae), micrococcus scarlatinae (Streptococcus pyogenes), or beta hemolytic streptococcus (β-haemolytic streptococci).

23. infecting, method according to claim 22, wherein said bacterium be selected from one or more in following: upper respiratory tract infection, lower respiratory infection, ear infection, pleura lung and bronchial infection, concurrent urinary tract infection, non-concurrent urinary tract infection, intra-abdominal infection, cardiovascular infection, bloodstream infection, septicemia, microbemia, CNS infects, Skin and soft tissue infection, GI infects, Arthropyosis infection, genital infection, ocular infection or granuloma infect, non-concurrent skin and skin texture infect (uSSSI), concurrent skin and skin texture infect (cSSSI), catheter infections, pharyngitis, sinusitis, external otitis, otitis media, bronchitis, pyothorax, pneumonia, Community-acquired bacterial pneumonia (CABP), Nosocomial Pneumonia (HAP), the Nosocomial bacterial pneumonia, ventilator associated pneumonia (VAP), infection in diabetic foot, vancomycin resistance faecalis infects, urocystitis and pyelonephritis, urinary stone disease, prostatitis, peritonitis, concurrent intra-abdominal infection (cIAI) and other intra-abdominal infection, the dialysis Related peritonitis, the internal organ abscess, endocarditis, myocarditis, pericarditis, the relevant septicemia of transfusion, meningitis, encephalitis, cerebral abscess, osteomyelitis, sacroiliitis, genital ulcer, urethritis, vaginitis, trachelitis, gingivitis, conjunctivitis, keratitis, endophthalmitis, infection in cystic fibrosis patient or heat generation neutrophilic granulocyte reduce disease patient's infection.

24. method according to claim 23, wherein bacterium infects and to be selected from one or more in following: Community-acquired bacterial pneumonia (CABP), Nosocomial Pneumonia (HAP), Nosocomial bacterial pneumonia, ventilator associated pneumonia (VAP), microbemia, infection in diabetic foot, catheter infections, non-concurrent skin and skin texture infect that (uSSSI), concurrent skin and skin texture infect (cSSSI), vancomycin resistance faecalis infects or osteomyelitis.

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